[Human chondrocyte responsiveness to bone morphogenetic protein-2 after their in vitro dedifferentiation: potential use of bone morphogenetic protein-2 for cartilage cell therapy]. / Réponse des chondrocytes humains à la bone morphogenetic protein-2 après leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage.

AIM OF THE STUDY: Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic. MATERIALS AND METHODS: Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored. RESULTS: Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages. CONCLUSION: The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.

Collagen-based biomaterials and cartilage engineering. Application to osteochondral defects.

Articular cartilage has a limited capacity for self-repair after trauma. Besides the conventional surgical techniques for repairing such defects, treatments involve implantation of autologous cells in suspension or within a variety of cell carrying scaffolds such as hyaluronic acid, alginate, agarose/alginate, fibrin or collagen. For the repair of full-thickness osteochondral defects, tissue engineers started to design single- or bi-phased scaffold constructs often containing hydroxyapatite-collagen composites, usually used as a bone substitute. The purpose of this study was to compare the behavior of bovine chondrocytes cultured in collagen-based scaffolds containing or not hydroxyapatite and cross-linked following two different methods. Calf chondrocytes seeded within Hemotèse and Collapat II sponges (SYMATESE biomaterials), chemically cross-linked with glutaraldehyde or EDC/NHS, were maintained up to one month in culture. The cells exhibited a similar behavior in the four scaffolds regarding proliferation level, deposition of glycosaminoglycans in the scaffolds and gene expression of types I, II and X collagens, aggrecan, MMP-1, -13 and the integrin subunits alpha10 and alpha11.

[Differentiation of adult human mesenchymal stem cells: chondrogenic effect of BMP-2]. / Différenciation des cellules souches mésenchymateuses adultes humaines: effet chondrogénique de la BMP-2.

Articular cartilage is essential for the motion of the skeleton. However, this tissue is unable to spontaneously repair once injured, since it is avascular and aneural. Numerous repair strategies are developed, but they do not lead to a functional tissue and research into cartilage repair focuses now on tissue engineering technics. Adult mesenchymal stem cells (MSC), present in various tissues, have the potential to differentiate into chondrocytes in vitro in response to specific growth factors. The members of the transforming growth factor beta, among them the bone morphogenetic protein (BMP)-2, appear very promising inducers in this context. BMP-2 favours chondrogenic expression, in particular expression of type IIB collagen, the cartilage-specific isoform of this collagen. Therefore, collagen type IIB is a good indicator of the differentiation state of MSC. However, since BMP-2 has also osteogenic properties, it is critical to differentially control chondrogenic and osteogenic properties of BMP-2 when used with MSC. Strategies for this control are presented in this review. Most likely, this is the combination of growth factors such as BMP-2 with biomaterials that will lead to the successful use of MSC for cartilage repair.

Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis.

OBJECTIVE: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs. METHODS: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and Ikappa-Balpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates. RESULTS: We selected a culture for 15 or 17 days with collagen sponges seeded with 10(7) chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha10 integrin sub-unit and low expression of COL1A2 and the alpha11 integrin sub-unit. In the presence of 1 ng/ml IL-1beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form. CONCLUSIONS: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs.

[Cartilage tissue engineering: state-of-the-art and future approaches]. / Ingénierie tissulaire du cartilage : état des lieux et perspectives.

Lesions of the articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Returning damaged cartilage in articular joints back to a functionally normal state has been a major challenge for orthopaedic surgeons. This interest results in large part because cartilage defects cannot adequately heal themselves. Current techniques used in orthopaedic practice to repair cartilage give variable and unpredictable results. Bone marrow stimulation techniques such as abrasion arthroplasty, drilling and microfracture produce mostly fibrocartilage. Autologous osteochondral transplant systems (mosaicplasty) have shown encouraging results. Autologous chondrocyte transplantation has led to a hyaline articular cartilage repair but little is known about the predictability and reliability of the procedure. The rapidly emerging field of tissue engineering promises creation of viable substitutes for failing cartilage tissue. Current tissue engineering approaches are mainly focused on the restoration of pathologically altered tissue structure based on the transplantation of cells in combination with supportive matrices and molecules. Among natural and synthetic matrices, collagen and polysaccharidic biomaterials have been extensively used with promising results. Recently, interest has switched to the use of mesenchymal stem cells instead of chondrocytes. Tissue engineering offers the possibility to treat localised cartilage lesions. Genetic engineering techniques using genetically modified chondrocytes offer also the opportunity to treat diffuse cartilage lesions occurring in osteoarthritis or inflammatory joint diseases. Electroporation is specially a reliable and inexpensive technique that shares with electrochemotherapy an ability to target the chondrocytes despite the barrier effect of the extracellular matrix without viral vectors. The authors review recent research achievements and highlight the potential clinical applications of new technologies in the treatment of patients with cartilage injuries.

Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges.

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.

Cartilage repair: surgical techniques and tissue engineering using polysaccharide- and collagen-based biomaterials.

Lesions of articular cartilage have a large variety of causes among which traumatic damage, osteoarthritis and osteochondritis dissecans are the most frequent. Replacement of articular defects in joints has assumed greater importance in recent years. This interest results in large part because cartilage defects cannot adequately heal themselves. Many techniques have been suggested over the last 30 years, but none allows the regeneration of the damaged cartilage, i.e. its replacement by a strictly identical tissue. In the first generation of techniques, relief of pain was the main concern, which could be provided by techniques in which cartilage was replaced by fibrocartilage. Disappointing results led investigators to focus on more appropriate bioregenerative approaches using transplantation of autologous cells into the lesion. Unfortunately, none of these approaches has provided a perfect final solution to the problem. The latest generation of techniques, currently in the developmental or preclinical stages, involve biomaterials for the repair of chondral or osteochondral lesions. Many of these scaffolds are designed to be seeded with chondrocytes or progenitor cells. Among natural and synthetic polymers, collagen- and polysaccharide-based biomaterials have been extensively used. For both these supports, studies have shown that chondrocytes maintain their phenotype when cultured in three dimensions. In both types of culture, a glycosaminoglycan-rich deposit is formed on the surface and in the inner region of the cultured cartilage, and type II collagen synthesis is also observed. Dynamic conditions can also improve the composition of such three-dimensional constructs. Many improvements are still required, however, in a number of key aspects that so far have received only scant attention. These aspects include: adhesion/integration of the graft with the adjacent native cartilage, cell-seeding with genetically-modified cell populations, biomaterials that can be implanted without open joint surgery and combined therapies, aimed at disease modification, pain relief and reduction of inflammation.

Influence of medium composition, static and stirred conditions on the proliferation of and matrix protein expression of bovine articular chondrocytes cultured in a 3-D collagen scaffold.

Interest in chemical and physical modifications of culture conditions and composition, as a way to improve engineered cartilage, has grown over the last decade. To address some of these aspects, articular bovine chondrocytes seeded in collagen sponges (2.3x10(6) cells/cm(3), whose growth and metabolism have been previously reported) were grown under static or stirred conditions (orbital shaker at 30 rpm), in either 10% FCS-supplemented or serum-free media (1% ITS+1mM cysteine). Under stirred conditions, we observed a 2-fold increase in both cell proliferation and sulphated glycosaminoglycan deposition after 1 month of culture, compared to static conditions, and after 3 months, a more homogeneous distribution of both cells and neomatrix in the constructs. During the first month of culture, the substitution of FCS by ITS led to low cell proliferation and poor neomatrix deposition but, after 2 months a steep increase was observed with ITS for these two parameters that reached, after 3 months the levels observed with FCS. Aggrecan was the more abundant component at both gene and protein levels, whereas the collagenous network formed was looser than with FCS. In conclusion, the use of these simple culture conditions should improve, in long-term culture, the quality of the cartilage construct.

Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering.

Collagen-based biomaterials in the form of sponges (bovine type I collagen, both native and cross-linked by treatment with diphenylphosphorylazide, noted control and DPPA sponges respectively) were tested as three-dimensional scaffolds to support chondrocyte proliferation with maintenance of the phenotype in order to form neocartilage. Control and DPPA sponges were initially seeded with 10(6) or 10(7) foetal bovine epiphyseal chondrocytes and maintained for 4 weeks in culture under static conditions in RPMI/NCTC medium with 10% FCS and without addition of fresh ascorbic acid. Both supports were always present during the study and a partial decrease in size and weight was detected only with control sponges, both seeded and unseeded. Cell proliferation was only noted in the 10(6) cells-seeded sponges (4-fold increase after 4 weeks of culture). Specific cartilage collagens (types II and XI) were deposited in the matrix throughout the culture and traces of type I collagen were noticed only in the culture medium after 2-3 weeks and 4 weeks in the case of 10(6) and 10(7) cells-seeded sponges, respectively. Glycosaminoglycans accumulated in the matrix, up to 1.8 and 9.8% of total dry weight after one month with both seeding conditions, which was much lower than in the natural tissue. In the 10(7) cells-seeded sponges, mineral deposition, observed with unseeded sponges, was significantly decreased (2- to 3-fold). These in vitro results indicate that both collagen matrices can support the development of tissue engineered cartilage.

Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B.

Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We studied the effects of ascorbic acid, retinoic acid (0.5 and 10 microM), and dihydrocytochalasin B (3, 10, 20 microM DHCB), separately or in combination (ascorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various agents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no induction of type I or X collagen synthesis nor increase in alkaline phosphatase activity. Chondrocytes treated with fresh ascorbic acid showed active protein synthesis associated with expression of types I and X after 6 and 13 days, respectively. This maturation was not accompanied by obvious hypertrophy of the cells or high alkaline phosphatase activity. Addition of retinoic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phosphatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 microM). DHCB was also a potent inhibitor of the maturation induced by treatment with ascorbic acid, as the chondrocytes maintained their rounded shape and synthesized type II collagen without induction of type I or X collagen. The pattern of protein secretion was compared under all culture conditions by two-dimensional gel electrophoresis. The different regulations of chondrocyte differentiation by ascorbic acid, retinoic acid, and DHCB were confirmed by the important qualitative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study.

Lineage-dependent collagen expression and assembly during osteogenic or chondrogenic differentiation of a mesoblastic cell line.

The mesoblastic clone, C1, behaves as a tripotential progenitor able to self-renew and to differentiate toward osteogenesis, chondrogenesis, or adipogenesis in response to specific inducers. In this study, expression and deposition by the C1 cells of essential components of the extracellular matrix, collagens type I, II, III, V, XI, VI, IX, and X were followed along the osteogenic and chondrogenic pathways, through biochemical, immunochemical, and electron microscopy analyses. Implementation of each program involves profiles of collagen synthesis and matrix assembly close to those documented in vivo. Depending on the applied inducers, cells adopt a defined identity and, controls acting at transcriptional and posttranslational levels adapt the set of deposited collagens to one particular cell fate. Osteogenic C1 cells selectively build a type I collagen matrix also containing type III, V, and XI collagens but selectively exclude type II collagen. Chondrogenic C1 cells first elaborate a type II collagen network and then acquire hypertrophic chondrocyte properties while assembling a type X collagen matrix as in the growth plate. This study provides an example of how a mesoblastic cell line can develop, in vitro, each of its genetic programs up to terminal differentiation. Intrinsic factors and time-dependent cell-matrix interactions might, as in vivo, underline the implementation of an entire differentiation program.

Analysis of types I, II, III, IX and XI collagens synthesized by fetal bovine chondrocytes in high-density culture.

OBJECTIVE: This study was undertaken in order to determine phenotypic modulation of the chondrocytes more closely in high-density culture conditions and to clarify the role of ascorbate. Levels of five collagen types were analyzed qualitatively and quantitatively, and their distribution was observed in the cell layer and the culture medium. DESIGN: Types I, II, III, IX and XI collagens, synthesized by fetal bovine chondrocytes in high-density culture, were analyzed qualitatively and quantitatively by direct measurement of radiolabeled collagens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by specific radioimmunoassays. RESULTS: Under the experimental conditions used in this study (0.6 x 10(6) cells/cm2), chondrocytes did not proliferate in the absence of ascorbate, whereas a twofold increase in cell number was observed in the presence of ascorbate at day 14. Cartilage-specific collagens (types II, IX and XI) were synthesized throughout the culture period (up to 47 days), as was type III collagen, which appeared as early as day 1 and was essentially present in the culture medium. Partial dedifferentiation of chondrocytes was demonstrated by the synthesis of type I collagen, which was detected by day 2 in culture medium containing ascorbate, and by day 6 without ascorbate. After 33 days of culture, a threefold increase in type I collagen synthesis was observed in culture medium with ascorbate, reaching 66% of the type II collagen content of the cell layer. One month of culture marked the onset of a progressive decrease in the synthesis of all collagen types. CONCLUSIONS: Under these high-density culture conditions, fetal bovine chondrocytes undergo a time and ascorbate-dependent program of partial dedifferentiation. This system provides a simple model for studying the initial mechanisms of chondrocytes dedifferentiation.

Two-dimensional electrophoresis of intracellular and secreted protein synthesized by fetal bovine chondrocytes in high-density culture.

In order to study the mechanisms involved in the differentiation/dedifferentiation of chondrocytes, fetal bovine chondrocytes in high-density cultures were treated with retinoic acid, an agent known to modify the chondrocyte phenotype (10 mumol/L between day 2 to day 5 of culture). The synthesis of intracellular and secreted proteins was studied by two-dimensional electrophoresis in cell lysates and culture media after labeling with [35S]methionine for the last 14 h of culture. The proteins expressed in control and retinoic acid-treated cells were identified by microsequencing after "in-gel" tryptic digestion of the spot or by immunodetection with specific antibodies after two-dimensional gel blotting. Intracellular protein modifications included one of 56.9 kDa and with an isoelectric point (pI) of 5.8 whose synthesis was previously reported to be up-regulated by 75%. Microsequencing of two internal peptides did not reveal a known protein. Changes to the chondrocyte phenotype were also recorded in the culture medium, as a decrease in type II collagen synthesis and expression of the small proteoglycan, decorin. Several new spots were also observed after treatment with retinoic acid, including a large, diffuse spot, not yet characterized, with a mean molecular mass of 39 kDa and a pI of 4.5-5.0. Under our experimental conditions, retinoic acid induces morphological changes of the chondrocytes and dramatic changes in the synthesis of several intracellular and secreted proteins that predate the synthesis of collagen type I (the classical marker of chondrocyte dedifferentiation).

Effect of retinoic acid on protein synthesis by foetal bovine chondrocytes in high-density culture: down-regulation of the glucose-regulated protein, GRP-78, and type II collagen.

The effect of 0.1-10 microM retinoic acid (RA) on foetal bovine chondrocytes was investigated in high-density cultures (0.6 x 10(6) cells/cm2). After 5 days of culture in ascorbate-free medium, control chondrocytes presented a typical rounded shape and synthesized type II, IX, XI and III collagens. After RA treatment on days 2-5 of culture, the cells exhibited a fibroblast-like shape and decreased synthesis of total protein (48%) and pepsinresistant proteins (60%) as determined by [35S]methionine labelling. Addition of RA was not followed by the expression of type I collagen, but induced quantitative changes in the synthesis of cartilage-specific collagens (II, IX and XI) as measured by direct autoradiography of the corresponding bands after SDS/PAGE. The main change was in type II collagen synthesis, with a 80% decrease in the cell-layer fraction and a 89% decrease in culture-medium fraction; inhibition of type IX and XI collagen synthesis was limited to 25 and 31% respectively. Modifications to intracellular proteins induced by RA were determined by using two-dimensional electrophoresis associated with a computerized imaging system. Synthesis of one of the more abundant proteins (pI 4.8; 78 kDa) was decreased by 75% after RA treatment. This protein was characterized by micro-sequencing as the glucose-regulated protein 78 (GRP 78). It was reported previously to bind denatured collagen and mutated type I procollagen molecule and to function as a molecular chaperone for collagen molecules. It remains to demonstrate whether the parallel down-regulation of GRP 78 and type II collagen observed here corresponds to a co-ordinate regulation of these two proteins.

Increased expression of tissue factor mRNA and procoagulant activity in ovine lentivirus-infected alveolar macrophages.

To link ovine lentivirus infection to lung tissue damage, we studied the procoagulant response in alveolar macrophages from experimentally infected lambs and in in vitro infected alveolar macrophages. We cloned ovine tissue factor cDNA and analysed its in vitro expression by Northern blotting. Visna-maedi virus induced tissue factor mRNA. In order to correlate this mRNA induction with its cellular function, we analysed macrophage procoagulant activity after in vitro and in vivo infection. The procoagulant activity was increased by interaction with the virus in both cases. Thus, visna-maedi virus-induced expression of tissue factor mRNA was associated with enhanced macrophage procoagulant activity. These findings indicate an active role of alveolar macrophages in the pathogenesis of these inflammatory lung lesions.

Dacron vascular biomaterial triggers macrophage ectoenzyme activity without change in cell membrane fluidity.

Biomaterials induce an inflammatory reaction characterized by a rapid recruitment at the implantation site of polymorphonuclear cells and macrophages. In the course of the inflammatory response, the cellular activation triggers expression of a number of enzymes, such as 5'-nucleotidase, which is widely distributed in animal cell membranes as an ectoenzyme. It is now well established that 5'-nucleotidase activity decreases following the contact of inflammatory cells with foreign particles. In this paper we investigate a possible correlation between the enzymatic activities and the dynamic properties of the cell membrane bilayer. Dacron pieces were introduced into rats' peritoneal cavities for a period of 6 h, after which the peritoneal cells were harvested, and various enzyme assays performed, including those for cytoplasmic, lysosomal, and ectoenzymes. In parallel, we studied cell membrane fluidity, using fluorescence polarization of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and cellular ultrastructural alteration resulting from the cell-biomaterial interactions using scanning and transmission electron microscopy. Our results show that: 1) macrophages spread around the Dacron fibers with cytoplasmic finger-like projections, but no phagolysosomes, 2) 5'-nucleotidase levels decrease with surgical trauma in comparison with the resident cell exudate, 3) implantation of biomaterials slightly modify the 5'-nucleotidase levels observed in the sham animal, 4) no differences in the anisotropy values indicating that membrane lipid order within the cells could not account for the observed decrease of 5'-nucleotidase activity. Thus, we can suggest that 5'-nucleotidase expression may reflect a particular feature of cell activation without a phagocytic process.

Evaluation of macrophage procoagulant and fibrinolytic activities induced by peritoneal dialysis solutions.

To investigate the coagulant and fibrinolytic potential of peritoneal macrophages, after short-term exposure to dialysis solutions intraperitoneal (IP) injection of these hyperosmolar glucose solutions was performed in rats. During the 72-hour postinjection period, the dialysis solutions and, as controls, Ringer's lactate and Ringer's lactate-glucose all induced a similar increase in the number of polymorphonuclear cells and macrophages within a maximum of 24 or 48 hours after their IP injection. These findings demonstrate that IP injection of any dialysis solution results in a moderate non-specific inflammatory cell harvesting. Compared with activity induced by the control solutions, no significant increase of procoagulant and fibrinolytic activities, identified respectively by the presence of thromboplastin and plasminogen activator, was observed in peritoneal macrophages obtained 48 hours after injection of the solution with the highest glucose concentration. However, the level of procoagulant activity could increase as a result of different manufacturers' processing of the solutions. That the basal level of macrophage functions may be modified suggests that this cell may initiate coagulolytic conditions in the peritoneal cavity, especially in the course of IP injection of dialysis solutions.

Peritoneal macrophage response: an in vivo model for the study of synthetic materials.

This study was designed to validate an in vivo model in rat to study cell-implant interactions. Pieces of Dacron and Goretex and polydimethylsiloxane (reference material) precoated or not were implanted in the peritoneal cavity. In some cases the peritoneal cells were stimulated before the implantation. Macrophage phagocytosis and fibrinocoagulolytic activities were investigated in parallel with morphological studies 6 h after implantation. A graded cell response related to the different situations was observed, providing a measure of the material's behaviour. Using this model, other investigations of macrophage/polymer can be conducted, especially to explain implant encapsulation.

Rat peritoneal macrophage procoagulant and fibrinolytic activities. An expression of the local inflammatory response.

1. Lysates of rat (Rattus norvegicus, Wistar strain) peritoneal macrophages selected by adherence were analysed for procoagulant activity (PCA) and plasminogen activator activity (PAA). 2. PCA is expressed following in vitro stimulation by lipopolysaccharide and in vivo (respectively 118 +/- 19.1 and 147.2 +/- 45.2 mUnits/10(6) M phi) very early after the intraperitoneal injection of thioglycollate. 3. PAA present in 24 hr thioglycollate stimulated cells (79.5 +/- 26.1 mI.U./10(6) M phi), disappears in a time dependent fashion after in vitro lipopolysaccharide stimulation. 4. Our findings indicate that rat peritoneal macrophages regulate their coagulolytic activities in a specific manner. 5. Rat peritoneal cavity may be proposed as a model for the study of inflammatory reaction with PCA and PAA as its indexes.