The effect of alginate, hyaluronate and hyaluronate derivatives biomaterials on synthesis of non-articular chondrocyte extracellular matrix.

Cartilage engineering consists of re-constructing functional cartilage by seeding chondrocytes in suitable biomaterials in vitro. The characteristics of neocartilage differ upon the type of biomaterial chosen. This study aims at determining the appropriate scaffold material for articular cartilage reconstruction using non articular chondrocytes harvested from rat sternum. For this purpose, the use of polysaccharide hydrogels such as alginate (AA) and hyaluronic acid (HA) was investigated. Several ratios of AA/HA were used as well as three derivatives obtained by chemical modification of HA (HA-C18, HA-C12(2.3), HA-C12(2.5)-TEG0.5). Sternal chondrocytes were successfully cultured in 3D alginate and alginate/HA scaffolds. HA retention in alginate beads was found to be higher in beads seeded with cells than in beads without cells. HA-C18 improved HA retention in beads but inhibited the chondrocyte synthesis process. Cell proliferation and metabolism were enhanced in all biomaterials when beads were mechanically agitated. Preliminary results have shown that the chondrocyte neo-synthesised matrix had acquired articular characteristics after 21 days culture.

Evaluation of cartilage repair tissue after biomaterial implantation in rat patella by using T2 mapping.

To evaluate the ability of MR T2 mapping (8.5 T) to characterize ex vivo longitudinally, morphologically and quantitatively, alginate-based tissue engineering in a rat model of patellar cartilage chondral focal defect. Calibrated rat patellar cartilage defects (1.3 mm) were created at day 0 (D0) and alginate sponge with (Sp/C+) or without (Sp/C-) autologous chondrocytes were implanted. Animals were sacrificed sequentially at D20, D40 and D60 after surgery and dissected patellae underwent MRI exploration (8.5 T). T2 values were calculated from eight SE images by using nonlinear least-squares curve fitting on a pixel-by-pixel basis (constant repetition time of 1.5 s, eight different echo times: 5.5, 7.5, 10.5, 12.5, 15.0, 20.0, 25.0 and 30.0 ms). On the T2 map, acquired in a transversal plane through the repair zone, global T2 values and zonal variation of T2 values of repair tissue were evaluated versus control group and compared with macroscopic score and histological studies (toluidine blue, sirius red and hematoxylin-eosin). "Partial", "total" and "hypertrophic" repair patterns were identified. At D40 and D60, Sp/C+ group was characterized by a higher proportion of "total" repair in comparison to Sp/C- group. At D60, the proportion of "hypertrophic" repair was two fold in Sp/C- group versus Sp/C+ group. As confirmed morphologically and histologically, the T2 map also permitted the distinction of three types of repair tissue: "total", "partial" and "hypertrophic". "Total" repair tissue was characterized by high T2 values versus normal cartilage (p<0.05). Zonal variation, reflecting the collagen network organization, appeared only at D60 for Sp/C+ group (p<0.05). "Hypertrophic" tissue, mainly observed at D60, presented high T2 global values without zonal variation with cartilage depth. These results confirm the potency of the MR T2 map (8.5 T) to characterize macroscopically and microscopically the patterns of the scaffold guided-tissue repair of a focal chondral lesion in the rat patella ("total", "partial" and "hypertrophic"). On T2 map, three parameters (i.e. MRI macroscopic pattern, T2 global values and zonal variation of T2 values) permit to characterize chondral repair tissue, as a virtual biopsy.

Expression of adhesion molecules and collagen on rat chondrocyte seeded into alginate and hyaluronate based 3D biosystems. Influence of mechanical stresses.

Chondrocytes use mechanical signals, via interactions with their environment, to synthesize an extracellular matrix capable to withstanding high loads. Most chondrocyte-matrix interactions are mediated via transmembrane receptors such as integrins or non-integrins receptors (i.e. annexin V and CD44). The aim of this study was to analyze, by flow cytometry, the adhesion molecules (alpha5/beta1 integrins and CD44) on rat chondrocytes seeded into 3D biosystem made of alginate and hyaluronate. These biosystems were submitted to mechanical stress by knocking the biosystems between them for 48 hours. The expression of type I and type II collagen was also evaluated. The results of the current study showed that mechanical stress induced an increase of type II collagen production and weak variations of alpha5/beta1 receptors expression no matter what biosystems. Moreover, our results indicated that hyaluronan receptor CD44 expression depends on extracellular matrix modifications. Thus, these receptors were activated by signals resulted from cell environment variations (HA addition and modifications owing to mechanical stress). It suggested that this kind of receptor play a crucial role in extracellular matrix homeostasis. Finally, on day 24, no dedifferentiation of chondrocytes was noted either in biosystems or under mechanical stress. For all biosystems, the neosynthesized matrix contained an important level of collagen, which was type II, whatever biosystems. In conclusion, it appeared that the cells, under mechanical stress, maintained their phenotype. In addition, it seems that, on rat chondrocytes, alpha5/beta1 integrins did not act as the main mechanoreceptor (as described for human chondrocytes). In return, hyaluronan receptor CD44 seems to be in relation with matrix composition.

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.

T2 mapping: an efficient MR quantitative technique to evaluate spontaneous cartilage repair in rat patella.

OBJECTIVE: To evaluate the ability of T2 mapping on an 8.5 T imager to characterize morphologically and quantitatively spontaneous repair of rat patellar cartilage following full thickness defect. METHODS: Patellar cartilage defects were created in 24 rats knees on D0. Eight rats per time-point were killed on D20, D40 and D60 after surgery. T2 maps of repair tissue in patellar defects were obtained from eight different axial spin echo images on an 8.5 T imager. Global, superficial and deep T2 values were evaluated in spontaneous repair tissues (3x8 right patellae) vs the opposite patellae (3x8 left patellae) of the same animals. MR data were compared with macroscopic and histological studies. RESULTS: T2 map was able to identify morphologically three types of repair tissue observed macroscopically and histologically: 'total', 'partial' and 'hypertrophic' repair tissue. 'Total' and 'partial' repair tissues were characterized by global T2 values almost similar to controls, whereas 'hypertrophic' repair tissues were characterized by T2 global values higher than controls. Zonal variation between superficial and deep T2 values observed in controls was not depicted in repair tissue before D60. CONCLUSION: T2 map is able to characterize quantitatively and qualitatively rat patellar cartilage repair, and thus can be promoted, as a non invasive technique, in clinical longitudinal studies of articular cartilage repair.

Endothelial cells grown on thin polyelectrolyte mutlilayered films: an evaluation of a new versatile surface modification.

Endothelial cell seeding constitutes an appreciated method to improve blood compatibility of small-diameter vascular grafts. In this study, we report the development of a simple innovative technique based on multilayered polyelectrolyte films as cell adhesive substrates. Polyelectrolyte multilayered films ending by poly(sodium-4-styrenesulfonate)/poly(allylamine hydrochloride) (PSS/PAH) or poly(L-glutamic acid)/poly(D-lysine) (PGA/PDL) could enhance cell adhesion by modification of the physico-chemical properties of the surface. The biological responses of human umbilical vein endothelial cells seeded on the polyelectrolyte multilayer films, on PDL or PAH monolayers, and on control surfaces, were evaluated in terms of initial attachment, growth, cellular metabolic activity, endothelial phenotype, and adhesion. The results showed that polyelectrolyte multilayers neither induce cytotoxic effects nor alter the phenotype of the endothelial cells. The polyelectrolyte multilayered films enhanced initial cell attachment as compared to the polyelectrolyte monolayer. Cell growth observed on the films was similar to that on TCPS. Among the different coating tested, the film ending by PSS/PAH exhibited an excellent cellular biocompatibility and appeared to be the most interesting surface in terms of cellular adhesion and growth. Such films could be used to cover hydrophobic (cell resistant) substrates in order to promote cell colonization, thereby constituting an excellent material for endothelial cell seeding.

Cartilage repair using new polysaccharidic biomaterials: macroscopic, histological and biochemical approaches in a rat model of cartilage defect.

OBJECTIVE: The present study aims at evaluating, in a rat model of cartilage defect, the potential of various polymers as filling and repair biomaterials. The macroscopic and histological observations are compared to biochemical parameters in order to appreciate the pertinence of the latter as suitable criteria in tissue engineering. METHODS: A hydrogel, consisting of hyaluronic acid (HA), covalently substituted by hydrophobic alkyl chains (HA12, HA18) and an alginate sponge, alone (Asp) or combined with HA (AHAsp) or combined with HA and chondrocytes (HYBsp) were evaluated. Cartilage lesions were drilled in femoral trochlea of rats. The analyses were performed on trochlea as well as on patella and condyles. RESULTS: Repairs achieved with hydrogels had a similar macroscopic appearance than those afforded by AHAsp and HYBsp. Best macroscopic and histological scores were obtained with HA18 and HYBsp in comparison with alginate group (P< 0.01 and P< 0.02 respectively). Biochemical evaluations confirmed the presence of similar amounts of proteoglycans in the repaired zones and in the controls, though with different DeltadiC4S/DeltadiC6S ratios and enhanced HA levels. CONCLUSIONS: Hydrogels or sponges proved to be colonized by cells synthesizing a matrix with a high HA content. The matrix obtained eventually turns hyaline and takes over the scaffold. The addition of HA and/or chondrocytes to Asp significantly improves the macroscopic and histological scores (P< 0.05 and P< 0.02 respectively). However, biochemical parameters are significantly different of those evaluated in native cartilage. The present study shows that only biochemical parameters allow to discriminate between various biomaterials in tissue engineering and are essential informations which should be taken into account in addition to macroscopic and histological observations.

In vitro study of intracellular IL-1beta production and beta1 integrins expression in stimulated chondrocytes--effect of rhein.

The purpose of the present study was to investigate the intracellular IL-1beta production and beta1 integrins (alpha4/beta1 and alpha5/beta1) expression on chondrocytes. Chondroytes monolayer (human chondrosarcoma cell line HEM-C55) were incubated for 12, 24 and 48 hours in the presence of Tumor Necrosis Factor-alpha (TNF-alpha, Sigma, France) or recombinant human IL-1alpha (rh-IL1alpha, Becton Dickinson, France). After direct immunolabelling, cells were either analyzed on FACScan flow cytometer (Becton Dickinson, France), or observed under an epi-fluorescence inverted microscope equipped with the CellScan EPR optical scanning acquisition system (IPLab-Scanalytics, USA). We found that the IL-1beta mean fluorescence intensity in flow cytometry and in 3D microscopy was increased in the presence of TNF-alpha or rh-IL-1alpha, and alpha4/beta1 or alpha5/beta1 expression was higher on stimulated cells than on control cells. On the other hand, we have evaluated the in vitro effects of rhein (10(-5) M, Negma, France), an active metabolite of diacerein, on the intracellular IL-1beta and beta1 integrins expressed by stimulated or no-stimulated chondrocytes. The results indicated that rhein leads to a reduction of IL-1beta synthesis whereas a weak decrease of beta1 integrins receptors expression is observed. From this study, it seems that rhein partially reduce cytokine-induced intracellular IL-1beta production, and it has a weak action on alpha4/beta1 or alpha5/beta1 receptors.

Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage.

Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix.

Amphiphilic derivatives of sodium alginate and hyaluronate for cartilage repair: rheological properties.

Various amphiphilic derivatives of sodium alginate and hyaluronate were prepared by covalent fixation of long alkyl chains (dodecyl and octadecyl) with various ratios on the polysaccharide backbones via ester functions. In the semidilute regime, aqueous solutions of the resulting compounds exhibited the typical rheological properties of hydrophobically associating polymers: tremendous enhancement of zero shear rate Newtonian viscosity, steep shear-thinning behavior, and formation of physically cross-linked gel-like networks. The influence of the alkyl chain length, its content on the polysaccharide and of the polymer concentration in the solution was well identified. All obtained results are discussed with respect to the schedule of conditions related to materials, which could be used for cartilage repair, such as in synovial fluid viscosupplementation as well as in cartilage replacement. In particular, it is seen that HA-C(12)-5 (hyaluronate substituted with 5% of dodecyl chains) and HA-C(18)-1 (hyaluronate substituted with 1% of octadecyl chains) in a 0.15N NaCl solution at 8 g/L have rheological properties quite similar to those of healthy synovial fluid. On the other hand, the rheological parameters of solutions at 8 g/L in 0.15N NaCl of some of derivatives, such as, for example, AA-C(12)-8 (alginate substituted with 8% of dodecyl chains) or HA-C(18)-2, are well fitted for a use in cartilage repair.

The role of 3D-microscopy in the study of chondrocyte-matrix interaction (alginate bead or sponge, rat femoral head cap, human osteoarthritic cartilage) and pharmacological application.

The potentialities of a new non-invasive optical scanning microscopy technique were evaluated through 3D analysis of chondrocyte-matrix interactions. Five different 2D or 3D culture systems were used: (1) MonoLayer (ML) of human chondrosarcoma cell line; (2) rat or human chondrocytes encapsulated in Alginate Bead (AB); (3) human chondrocytes encapsulated in Alginate Sponge (AS); (4) Rat Femoral Head Cap (RFHC); (5) slices of knee human Osteoarthritic Cartilage (HOAC). Chondrocytes ML, AB, RFHC were incubated for 24 h in vitro in the presence of recombinant human interleukin1-beta (rhIL1-beta) and the effects on cytoskeleton organisation (F-actin filament), Focal Adhesion Kinase (FAK) expression (tyrosine kinase), collagenase B expression (metalloprotease) were studied. Furthermore, the production of intracellular IL1-beta by LPS- or rhIL1-beta-stimulated chondrocytes was shown to be partly suppressed by rhein (active metabolite of diacerhein) in all culture systems. This high resolution light microscopy gave complementary information that could be important for a better understanding of the interaction of chondrocytes with the extracellular matrix in a variety of culture devices.

High interaction alginate-hyaluronate associations by hyaluronate deacetylation for the preparation of efficient biomaterials.

The paper presents fundamental investigations of alginate-hyaluronate association with significant polymer interactions for preparation of efficient biomaterials. For this purpose, acetamide functions of hyaluronate were partly cleaved by hydrazine at high temperature, yielding amino groups accessible to carboxylic functions of the alginate chain. Alginate-hyaluronate association was studied both in dissolved state by rheological measurements and CD, and in the form of gel slabs prepared after calcium diffusion. Appreciable interaction between carboxylic groups of alginate and the released amino groups of hyaluronate was put into evidence by enhanced values of the viscosity of mixed solutions, and by assessment of the properties of the gel formed: moderate deacetylation allowed gels of improved hardness and viscosity. Nevertheless, high deacetylation was observed to hinder the gel formation by Ca(2+) complexation of alginate, by the significant competition of COOH-NH(2) association. Interaction between alginate and modified hyaluronate results in regular gel structure, with small cavities.

Hyaluronate-alginate gel as a novel biomaterial: mechanical properties and formation mechanism.

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated to combine the gel-forming properties of alginate with the healing properties of hyaluronate. Gels were prepared by diffusion of calcium into alginate-hyaluronate mixtures, with an alginate content of 20 mg/mL. The hyaluronate source was shown to have significant effect on the aspect and the properties of the gels. The gels have viscoelastic behaviour and the transient measurements carried out in creep mode could be interpreted through a Kelvin-Voigt generalised model: experimental data led to the steady state hardness and a characteristic viscosity of the gel. Gels prepared from Na rooster comb hyaluronate with weight ratio up to 0.50 have satisfactory mechanical properties, and fully stable gels are obtained after a few days; on the contrary, use of lower molecular weight hyaluronate led to loose gels for hyaluronate contents over 0.25. Gel formation was investigated by measurements of the exchange fluxes between the calcium chloride solution and the forming gel, which allowed thorough investigations of the occuring diffusion phenomena of water, calcium ion and hyaluronate. Strong interactions of water with hyaluronate reduce significantly the rate of weight loss from the gel beads and allows higher water content in steady-state gels. Calcium content in the gel samples could be correlated to the actual alginate concentration, whatever the nature and the weight ratio of hyaluronate.

Hyaluronate-alginate combination for the preparation of new biomaterials: investigation of the behaviour in aqueous solutions.

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated. Crossed techniques were used to assess the existence of polymer interactions in aqueous solutions up to 20 mg/ml. Alginate was obtained from algae and hyaluronate was purified from rooster comb. Viscometry measurements using the capillary technique or the Couette flow, together with circular dichroism investigations, evidenced the moderate significance of interactions between the two polysaccharides in dilute solutions. In addition, the case of more concentrated solutions and containing 20 mg/ml alginate was approached by rheological measurements in the flow mode; the behaviour of the polymer associations appeared as a compromise between those of individual polysaccharides.

Separation and quantification by ion-association capillary zone electrophoresis of unsaturated disaccharide units of chondroitin sulfates and oligosaccharides derived from hyaluronan.

An analytical method has been developed for assay of unsaturated disaccharides of chondroitin sulfates and of oligosaccharides (tetra- and hexasaccharides) of hyaluronan, using ion-association capillary zone electrophoresis. Samples were applied at the anode (the usual polarity), using a borate buffer modified by an ion-pairing reagent, tetrabutylammonium (TBA) phosphate, and the effect of the concentration of the ion-pairing reagent on various electrophoretic parameters (electroosmotic flow, electrophoretic mobility of products, capacity factors) was observed. Increasing concentrations of the reagent led to a decrease of zeta potential, probably due to specific adsorption of the quaternary ammonium ion onto the capillary wall. The authors propose a mechanism of separation, in which anionic borate complexes are formed and interact with TBA ion inside the capillary tube. The capillary electrophoretic system described is potentially a powerful method for specific measurement of the concentrations in joint tissues of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronan, whose relative abundances may vary in various diseases or after local treatments with, for example, antiinflammatory drugs, chondroprotective agents, or orthopedic implants.

Cytocompatibility of Ti-6Al-4V and Ti-5Al-2.5Fe alloys according to three surface treatments, using human fibroblasts and osteoblasts.

Titanium alloys are well known for their superior mechanical properties as well as for their good biocompatibility, making them desirable as surgical implant materials. However, these alloys have been proven to behave poorly in friction since wear particles were often detected in tissues and organs associated with titanium implants. In this paper, three surface treatments were investigated in order to improve the wear resistance and the hardness of Ti-6Al-4V and Ti-5Al-2.5Fe: (a) glow discharge nitrogen implantation (10(17) atoms cm-2), (b) plasma nitriding by plasma diffusion treatment (PDT) and (c) deposition of TiN layer by plasma-assisted chemical vapour deposition (PACVD) additionally to PDT. Surface characterization after the different treatments showed considerable improvement in surface hardness, especially after the two nitriding processes. Moreover, the good corrosion resistance of untreated alloys was maintained. A cell culture model using human cells was chosen to study the effect of such treatments on the cytocompatibility of these materials. The results showed that Ti-5Al-2.5Fe alloy was as cytocompatible as the Ti-6Al-4V alloy and the same surface treatment led to identical biological consequences on both alloys. Nitrogen implantation did not modify at all the cellular behaviour observed on untreated samples. After the two nitriding treatments, cell proliferation and viability appeared to be significantly reduced and the scanning electron microscopy study revealed somewhat irregular surface states. However, osteoblast phenotype expression and protein synthesis capacity were not affected. PDT and PACVD may be interesting alternatives to the physical vapour deposition technique.

Evaluation of the effect of three surface treatments on the biocompatibility of 316L stainless steel using human differentiated cells.

AISI 316L stainless steel (SS) is widely used in orthopaedic implantology, although biological complications may result from its insufficient mechanical and tribological properties. In order to improve the wear and corrosion resistance as well as the hardness of 316L SS, three surface treatments, derived from those applied in mechanical engineering industries, were investigated: (1) glow discharge nitrogen implantation, (2) carbon-doped stainless steel coating sputtering and (3) low temperature plasma nitriding. Surface characterization according to the different heat treatments showed that corrosion and wear resistance were strongly improved, especially by ion implantation or carbon-doped SS coating sputtering. In the same way, microhardness was significantly increased after the three treatments. The effect of such treatments on the biocompatibility of 316L SS was studied with human osteoblast and fibroblast cultures. Basic and specific features of the cells showed that ion-implanted and carbon-doped stainless steels were biocompatible, whereas dramatic cellular reactions were noted when contacted with nitrided stainless steel. A hypothesis is given to explain this observation but further experiments are needed to optimize the nitriding process. Nitrogen implantation and carbon-doped layer deposition could be efficient means for improving the physical properties of stainless steel without affecting its biocompatibility. Such surface treatments may have relevance for increasing the life time of 316L biomedical devices.

Stereoselective distribution of tiaprofenic acid in synovium and cartilage in osteoarthritic patients.

OBJECTIVE: In order to document the stereoselective distribution in joints of a chiral nonsteroidal anti-inflammatory drug, the relative affinities of the enantiomers of tiaprofenic acid in synovium and for cartilage were compared. METHODS: The distribution of tiaprofenic acid in synovium and in cartilage was studied 25 h after administering the racemic drug for 2 days (600 mg of a sustained-release preparation, once daily), in 12 inpatients with osteoarthritis of the hip requiring arthroplasty. Enantiomers were quantified in plasma and freeze-ground tissues by a chiral HPLC assay. RESULTS: Plasma concentrations of the dextrorotatory (R) enantiomer (0.40 microgram/ml) were higher than those of its antipode. The concentration of racemate in synovium (in dried and fresh tissues, 150% and 40%, respectively, of the concentration in plasma) was much higher than that in cartilage (in dried tissues 32% of the plasma concentration). The ratio of the active, dextrorotatory (R) enantiomer to its antipode was higher in synovial tissue than in plasma. CONCLUSION: Tiaprofenic acid is distributed stereoselectively in plasma and synovium, which contain a higher concentration of the active, dextrorotatory (R) enantiomer. In cartilage, it reaches only a very low concentration.