Ion exchanges in apatites for biomedical application.

The modification of the composition of apatite materials can be made by several processes corresponding to ion exchange reactions which can conveniently be adapted to current coatings and ceramics and are an alternative to setting up of new synthesis methods. In addition to high temperature thermal treatments, which can partly or almost totally replace the monovalent OH- anion of stoichiometric hydroxyapatite by any halogen ion or carbonate, aqueous processes corresponding to dissolution-reprecipitation reactions have also been proposed and used. However, the most interesting possibilities are provided by aqueous ion exchange reactions involving nanocrystalline apatites. These apatites are characterised by the existence on the crystal surface of a hydrated layer of loosely bound mineral ions which can be easily exchanged in solution. This layer offers a possibility to trap mineral ions and possibly active molecules which can modify the apatite properties. Such processes are involved in mineralised tissues and could be used in biomaterials for the release of active mineral species.

Copper modulation of extracellular matrix synthesis by human articular chondrocytes.

OBJECTIVE: The effects of Cu2+ on human articular chondrocytes, arising from both N (normal) and OA (osteoarthritic) cartilage, were investigated "in vitro". METHODS: Chondrocytes, cultured in high density, were incubated with copper chloride (0.01-0.25 microM/mL). Proteoglycan and collagen were assessed by incorporation of [35S]-Sulfate and [3H]-Proline. SDS-PAGE analysis was performed to quantify the ratio of type II to type I collagen. RESULTS: Cu2+ neither increased proteoglycan synthesis by chondrocytes. of origin N or OA, nor influenced their proliferation rate. Collagen synthesis was increased. This effect is time and concentration dependant: in cultures treated for 12 days, collagen synthesis stimulation was +20% and +26% (P < 0.02) in N and OA cultures respectively, the ratio of type II to type I collagen was slightly increased. This effect was more obvious in OA cell lines than in N ones. CONCLUSION: The observations suggest that Cu2+ upregulates collagen anabolism in human articular chondrocytes.

In vitro kinetic evaluation of titanium alloy biodegradation.

The present controlled in vitro experiment evaluated the dissolution kinetics of titanium (Ti), aluminum (Al) and vanadium (V). Titanium alloy (Ti90Al6V4) dental implants were inserted in 1.8 ml sterile tubes, containing equal volumes of NaCl 0.9% (w/v) and human serum. Metallic elements released by the atomic process of corrosion were measured at pH 7.2 and 37 C by atomic absorption spectrophotometer at 1, 3, 6, 9, 15, 21, 27, 33, 42, 51, 60, 69, 78, 87 and 96 days. Ti dissolution averaged 16+/-5 ng/cm2/day and 1565 ng/cm2 over the experimental period. Al dissolution was stable at 9+/-5 ng/cm2/day and averaged 945 ng/cm2 over the 96-day period. V dissolution was stable at 0.15+/-0.18 ng/cm2/day after the sixth day of incubation and averaged 42 ng/cm2 over the 96-day period. Major disparities in atomic dissolution were detected among implants. No local or systemic reaction to titanium has been documented. In contrast, 4% toxic V and 6% Al may suffice to elicit local and systemic reactions or inhibit cellular proliferation and differentiation.

Apoptosis in normal and osteoarthritic human articular cartilage.

OBJECTIVES: To investigate whether apoptosis occurs in osteoarthritis (OA), and if this phenomenon is modulated by human recombinant interleukin 1beta (hrIL1beta). METHODS: Human articular cartilage samples were obtained at the time of hip arthroplasty because of femoral neck fracture (normal cartilage) (n=4) or advanced coxarthrosis (OA cartilage) (n=14). Apoptotic chondrocytes, isolated by collagenase digestion and cultivated for 24 hours, or present in situ in frozen cartilage sections, were quantified by fluorescent microscopy using two apoptosis markers: the TUNEL reaction, which detects nuclear DNA fragmentation, and Annexin-V-fluos, which labels at the membrane level the externalisation of phosphatidylserine. RESULTS: In OA cartilage 18-21% of chondrocytes showed apoptotic features, compared with 2-5% in normal cartilage. The results were similar for the two comparative studies (in situ and in vitro) and for both apoptosis markers. Moreover, hrIL1beta increased the apoptosis rate in vitro in a dose dependent manner in OA and normal chondrocytes. CONCLUSION: These results suggest that apoptosis may be an important factor in the evolution of OA and may be a new target for treatment of OA.

In vitro comparative evaluation under static conditions of the hemocompatibility of four types of tubing for cardiopulmonary bypass.

A comparative in vitro assessment of 4 types of tubing representative of the materials currently used in cardiopulmonary bypass (CPB) procedures was conducted under static conditions using liquid extracts of the materials or direct contact with fresh human blood or serum. The parameters monitored were biomarkers of coagulation and fibrinolytic cascades, the complement system and cell activation. Silicone and PVC tubing were shown to be non-cytotoxic and non-hemolytic. Heparin-coated PVC tubing did present a certain degree of cytotoxicity especially when in direct contact. Thrombosis was found to be significantly lower with the same heparin-coated material. To a lesser extent, platinum-cured silicone also showed a reduced thrombotic tendency. None of the materials activated platelets or the complement system. With platinum-cured silicone tubing, constant and lower leukocyte adhesion was evidenced at the different experimental time points. This could reflect reduced cell activation.

The effect of recombinant human basic fibroblast growth factor rhFGF-2 on human osteoblast in growth and phenotype expression.

This paper describes the studies of human recombinant basic fibroblast growth factor (rhFGF-2) for its effects on human osteoblast growth and phenotype expression. During a 24-h period of treatment, rhFGF-2 highly stimulated DNA synthesis in a dose-related fashion with a maximum stimulation of 150% for 1 ng/ml. On the other hand, rhFGF-2 decreases alkaline phosphatase activity, synthesis of type I collagen, and cumulative amount of osteocalcin. Moreover, rhFGF-2 provoked a threefold increase in the amount of intracellular cAMP. Scatchard plots show the presence of two classes of [125I] rhFGF-2 receptors. This data suggests that rhFGF-2 which stimulate cell replication may act indirectly as an anabolic agent and stimulate some of the phenotypic expression markers.

In vitro study of biodegradation of a Co-Cr alloy using a human cell culture model.

The evaluation of a potential biomaterial is based on two approaches: firstly, the study of the local and systemic effects of the biomaterial implanted in the host; and secondly the study of the behaviour of the biomaterial itself with increasing time. The progress achieved in human cell culturing allows in vitro evaluation of a new biomaterial using the human cell(s) system(s) characteristic of the tissue which it will be exposed to in vivo. This kind of approach permits the assessment of the biodegradation of a biomaterial whatever it is: metal; alloy; ceramic; glass; polymer; with or without specialized coating.... The experimental approach is as follows: discs representative of the biomaterial (surface state, cleaning, sterilization process) are manufactured in order to cover the bottom of the culture wells. Thereafter, they are either brought in the presence of complete culture medium alone, or in the presence of a subconfluent cell layer. A kinetic analysis is performed using various incubation periods at 37 degrees C. Released biodegradation products are identified and quantified, in both the medium and cell compartment, and on the other hand cytotoxicity is assessed. A Co-Cr alloy was studied over a 9-day period according to the experimental schedule, and showed a higher corrosion rate in the presence of osteoblasts in the range of 25-30%. Moreover, an intracellular uptake of both Cr and Co was detected, which will have physiological importance.

Osteogenin (bone morphogenic protein 3) inhibits proliferation and stimulates differentiation of osteoprogenitors in human bone marrow.

Treatment of human bone marrow osteoprogenitors with osteogenin (BMP-3; at 1, 2.5 and 10 ng/ml) caused dose- and time-dependent inhibition of DNA synthesis and cell proliferation. Simultaneously, osteogenin stimulated type I collagen synthesis and cAMP production. Addition of osteogenin to the cell culture increased intracellular alkaline phosphatase activity and osteocalcin synthesis, with maximal stimulation at 2.5 ng/ml. Simultaneous addition of 2.5 ng/ml osteogenin and 1,25 dihydroxy vitamin D3 (10(-8) M) enhanced the stimulation observed in osteocalcin synthesis. The experiments reported here demonstrate the significant "in vitro" influence of osteogenin in the stimulation of osteogenic phenotype in osteoprogenitor cells which have been isolated from human bone marrow and cloned. These results support a reciprocal relationship between cell growth inhibition and expression of osteoblast differentiation.

Effect of a derivatized dextran on human osteoblast growth and phenotype expression.

Water soluble derivatized dextran named E9 with a molecular weight of 45,000 g l-1 containing 58% methyl carboxylic acid unit, 19% benzylamide unit, and 26% sulfonate with a specific anticoagulant activity of 0.29 IU mg-1 was studied for its effects on human osteoblast growth and phenotype expression for short-term treatment. At concentrations between 1 ng ml-1 and 1 microgram ml-1 E9 has no effect on DNA synthesis whereas at higher concentrations DNA synthesis is inhibited in a dose related fashion (87% for 400 micrograms ml-1). For concentrations which do not modify osteoblast growth, E9 promotes alkaline phosphatase activity, type I collagen and osteocalcin synthesis with a maximum effect for 0.1-1 microgram ml-1. It has a synergistic effect with hPTH increasing AMPc. Moreover, osteonectin synthesis was enhanced in a dose-dependent manner between 0.1 and 5 micrograms ml-1. These results seem to indicate that E9 is able to stimulate human osteoblast phenotype expression and could be useful in clinical applications.

Radioimmunodetection of rat and rabbit cartilage using a monoclonal antibody specific to link proteins.

Biodistribution analysis using [125I]Fab-6F3 specific to link proteins from human articular cartilage performed in rats by autoradiography showed a high concentration of radioactivity in all cartilaginous tissues. Preliminary immunoscintigraphic assays were performed in rabbits. Front and side view images of whole animals exhibited high uptake in cartilage tissue of the knee articulation, in the invertebral disk and the humeral head. This fixation was still detected 24 h post-injection, although high washout of radioactivity was observed.

Molecular interface characterization in human bone matrix. I. Biochemical and IR spectroscopic studies.

The aim of this work is the characterization of interfaces in calcified tissues. In order to separate the non-collagenous bone proteins, according to their interaction with collagen or hydroxyapatite crystals, 10 sequential bone demineralizations using EDTA alone were carried out, followed by four sequential extractions using both EDTA and GuHCl. The extracts were characterized by SDS-PAGE, IR spectrum analysis, and kinetics of demineralization and proteins released. A great proportion of non-collagenous proteins are bound to the collagen matrix, many of which have a high affinity to it. This work demonstrates that collagen is not directly linked to the mineralized phase.

Human bone marrow stromal cells express an osteoblastic phenotype in culture.

This study reports the selection and characterization of osteogenic precursors from human bone marrow which were isolated by two "clonings" and successive subculturing. These cell lines express alkaline phosphatase activity. Gel electrophoresis of [3H]-proline labeled cultures showed that the cloned cells produce only type I collagen. They synthetize osteocalcin and osteonectin. They respond to 1,25 dihydroxy vitamin D3 by increasing osteocalcin synthesis and secretion, and to parathyroid hormone by increasing cyclic AMP synthesis. After the third subculture in the absence of beta-glycerophosphate, these cell lines formed lots of clusters which exhibit high alkaline phosphatase activity and positive von Kossa staining. X-ray energy spectrum shows that these cells are surrounded by "budding" structures containing calcium and phosphorus with a ratio Ca:P identical to those of pure hydroxyapatite. This process was associated with 45Ca uptake into the cells. All these data support the selection of osteogenic cells which may be of considerable clinical importance.

Production of epidermal sheets in a serum free culture system: a further appraisal of the role of extracellular calcium.

Rhenwald and Green's technique is currently the standard method for growing stratifying epidermal cell cultures. The serum free system developed in Ham's laboratory (MCDB 153) was designed to grow keratinocyte monolayers in clonogenic conditions. Our aim was to optimize conditions in serum-free MCDB 153 for culturing epidermal sheets from adult normal skin, and to assess the effect of extracellular calcium and temperature on proliferation and differentiation of cultured keratinocytes. Sixteen strains derived from plastic surgery specimens (mean age of donors 37 years; range 5-89) were used. Primary cultures were seeded at an optimal density of 8 x 10(4) cells/cm2 in primary cultures and 10(4) cells/cm2 in secondary cultures in complete medium including EGF, insulin, hydrocortisone and bovine pituitary extract, supplemented with isoleucine, tyrosine, methionine, phenylalanine, tryptophane and histidine. Amino acid (AA) supplementation allows a 5.8-fold increase in cell counts at confluency and monolayers with densely packed cells are obtained. In AA supplemented cultures, confluency is obtained in 16 +/- 3 days in primary cultures and in 13 +/- 0.5 days at first passages. Switches to 1.1 mM calcium at first or second passages resulted in a significant increase in cell counts (P less than 0.001), when compared with AA supplemented low calcium cultures. Low temperature/low calcium cultures resulted in a 50% decrease in cell counts. Low temperature/high calcium cultures gave similar cell counts as the 37 degrees C controls. AA and calcium supplemented cultures were evaluated for differentiation markers: involucrin expression was increased, keratins 5, 6, 14, 17 were expressed, and the sheets were 6-10 layers thick by electron microscopy, with keratohyalin granules and cornified envelopes appearing at layers 3-6 (from basal layer). Dispase treatment allowed an easy detachment of these sheets. These results show that the culture medium MCDB 153 can be adapted without serum supplementation to batch culture of human adult keratinocytes to produce epidermal sheets suitable for grafting. They also indicate that extracellular calcium in physiological range of concentration is not a sufficient signal for growth arrest when other growth conditions are optimized.

Cytocompatibility of two coating materials, amorphous alumina and silicon carbide, using human differentiated cell cultures.

The cytocompatibility of two coating materials, amorphous alumina and silicon carbide deposited by radio-frequency sputtering, was studied using alveolar bone osteoblasts and gingival fibroblasts from human healthy tissues. Cytocompatibility was assessed at the level of both the basic (attachment, proliferation and cell protein content) and the specific features (intracellular alkaline phosphatase activity and the cytoskeleton) of the cells in direct contact with the coating. Titanium was used as the reference material. The results showed that both silicon carbide and amorphous alumina are cytocompatible for human fibroblasts and osteoblasts, whereas titanium appears the least cytocompatible of all the three substrates. Moreover, the amorphous alumina coating seems slightly bioactive. It seems that these coatings, particularly amorphous alumina, could be used to protect alloys against corrosion, and consequently combine the good mechanical properties of the alloys with the good biocompatibility of the coatings. These coatings seem to perform more suitably than titanium if the strength of the bond between the coating and the underlying alloys is strong enough to give a stable composite material.

In vitro evaluation of an epoxy resin's cytocompatibility using cell lines and human differentiated cells.

The cytocompatibility of a polyepoxy resin (Elf Aquitaine) has been studied using both cell lines and human differentiated cell cultures. The human models were gingival fibroblasts and bone osteoblasts, while the cell lines were Hela cells and 3T3 Balb/c cells. Basal cytocompatibility was assessed by estimation of the cell proliferation, total cell protein content, cell membrane sub-lysis, and cell attachment and spreading. Specific cytocompatibility concerning human osteoblasts, from both alveolar and trabecular bone, was determined by measuring the intracellular alkaline phosphatase activity. Resin colonization by the cells was studied by both TEM and SEM. The behaviour of the two cell lines reveals a significant level of discrepancy, whereas the behaviour of human cells, whatever the model, is comparable; however, osteoblasts look more sensitive. Moreover, the results show that this epoxy resin exhibits a moderate cytocompatibility which could be the result of the cytotoxicity of early released products, associated with the considerable surface roughness.

Biodistribution study of a monoclonal antibody specific to link proteins from human cartilage.

We report the use of monoclonal antibody 6F3 prepared against link proteins from human articular cartilage to elucidate the distribution of these glycoproteins within connective and other tissues. By immunohistochemical analysis, we showed that only the Fab fragment could reach the antigenic site in human articular sections. Cross-reactivity of the antibody 6F3 with link proteins purified from rat articular cartilage allowed us to carry out a biodistribution analysis in vivo in the rat. The time course of whole blood and plasma showed maximal activity 6 h after the 20 micrograms i.v. injection of [125I]Fab-6F3. Urinary excretion seems to be a high route of elimination. Moreover, we noticed no radioactive uptake across the blood-brain barrier. A significant fixation of labeled antibody Fab-6F3 was observed in noncartilaginous connective tissues such as aorta, skin and lung. As expected, specific and increased radioactivity was observed in all cartilage tissues, this increase was significantly higher 6 h after the [125I]Fab-6F3 injection than in the other connective tissues.

Fibrin seal in wound healing: effect of thrombin and [Ca2+] on human skin fibroblast growth and collagen production.

To assess the interaction of a fibrin sealant with wound healing, an in vitro study using human skin fibroblasts was carried out. The effect of thrombin and calcium concentrations in the sealant on the growth parameters and collagen synthesis by normal human skin fibroblasts was examined. The fibroblast proliferation was increased 3 times for 50 and 25 IU of thrombin/ml. However for 20 mM [Ca2+], this stimulating effect of thrombin was observed after an 8 day incubation period, whereas it was observed as soon as the 2nd day in the presence of 2 mM [Ca2+]. The high rate of [Ca2+] (20 mM) partly inhibited DNA synthesis: for 2 mM [Ca2+], the incorporation of [3H]-Thymidine was 4 times greater than for 20 mM [Ca2+]. Further experiments demonstrated that human skin fibroblasts in the presence of 50 IU of thrombin/ml and 2 mM [Ca2+] in fibrin seal could increase the type I and III collagen synthesis while increasing the ratio of type III to type I. Thus, our results suggest that in vivo wound healing which required fibroblast growth and collagen synthesis can be stimulated in the presence of fibrin glue which is in good accordance with previous clinical observations.

Study of the effect of the surface state on the cytocompatibility of a Co-Cr alloy using human osteoblasts and fibroblasts.

Cobalt-chromium-based alloys are widely used in oral and orthopedic implantology. Although they are relatively well tolerated, biological complications could occur which sometimes are due to the insufficient biocompatibility of the alloy. This study shows the effects of an alloy (Co (base), 28% Cr, 5.5% Mo, 1% Ni, 0.95% Si, 0.7% Fe, 0.65% Mn, 0.25% C), on differentiated human cells derived from an oral implantation site, specifically alveolar bone osteoblasts and gingival fibroblasts. The cytocompatibility of the alloy is determined by the study of cell proliferation, determination of total cell protein and intracellular alkaline phosphatase contents, cytoskeleton, and cell morphology. The alloy is presented to the cells in four different surface states: rough cast, specular polished, microbead blasted, and RF sputtered. The results demonstrate that the same material has different effects on the basal and specific cellular functions, according to its surface state. For this alloy we can classify its cytocompatibility according to its surface state in such an order: Microbead blasted much greater than specular polished greater than RF sputtered greater than rough cast.