Seaweed polysaccharide-based hydrogels used for the regeneration of articular cartilage

This manuscript provides an overview of the in vitro and in vivo studies reported in the literature focusing on seaweed polysaccharides based hydrogels that have been proposed for applications in regenerative medicine, particularly, in the field of cartilage tissue engineering. For a better understanding of the main requisites for these specific applications, the main aspects of the native cartilage structure, as well as recognized diseases that affect this tissue are briefly described. Current available treatments are also presented to emphasize the need for alternative techniques. The following part of this review is centered on the description of the general characteristics of algae polysaccharides, as well as relevant properties required for designing hydrogels for cartilage tissue engineering purposes. An in-depth overview of the most well known seaweed polysaccharide, namely agarose, alginate, carrageenan and ulvan biopolymeric gels, that have been proposed for engineering cartilage is also provided. Finally, this review describes and summarizes the translational aspect for the clinical application of alternative systems emphasizing the importance of cryopreservation and the commercial products currently available for cartilage treatment.Authors report no declarations of interest. Authors thank the Portuguese Foundation for Science and Technology (FCT) for the PhD fellowship of Elena G. Popa (SFRH/BD/64070/2009) and research project (MIT/ECE/0047/2009). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS

[Specific measurement of alternate pathway activation of complement in human glomerulonephritides (GN): 125 cases (author's transl)]

International audienceC3 cleaving activity through alternate pathway, appreciated by native C3 antigen reduction in the presence of Mg EGTA, has been measured in sera of 40 controls and of 125 GN patients. The normal percentage of native C3 conversion ranged from 0 to 29 in controls (p less than 0.05). The number of positive samples is 0/30 in lupus GN (0%), 13/29 in acute GN (45%) and 26/66 in membranoproliferative GN (39%) with 7/8 in dense deposits subtype. The presence of alternate pathway activators of complement correlated with serum C3 level: their frequency is respectively 0, 0.32, and 0.48 if serum C3 is normal, low, and very low. Such activity correlated also well with alternate pathway C3 activation (low C3 and normal C4): the frequency of positive samples is respectively 0, 0.07, and 0.51 in the presence of No C3 activation, classical pathway activation (low C3 and low C4), and alternate pathway activation. The nature of such serum activators could be nephritic factor, bacterial polysaccharides, polymeric IgA, activated properdin, peculiar immune complexes, or other factors. Such measurement is an improvement immunopathological step in the investigation of human GN.C3 cleaving activity through alternate pathway, appreciated by native C3 antigen reduction in the presence of Mg EGTA, has been measured in sera of 40 controls and of 125 GN patients. The normal percentage of native C3 conversion ranged from 0 to 29 in controls (p less than 0.05). The number of positive samples is 0/30 in lupus GN (0%), 13/29 in acute GN (45%) and 26/66 in membranoproliferative GN (39%) with 7/8 in dense deposits subtype. The presence of alternate pathway activators of complement correlated with serum C3 level: their frequency is respectively 0, 0.32, and 0.48 if serum C3 is normal, low, and very low. Such activity correlated also well with alternate pathway C3 activation (low C3 and normal C4): the frequency of positive samples is respectively 0, 0.07, and 0.51 in the presence of No C3 activation, classical pathway activation (low C3 and low C4), and alternate pathway activation. The nature of such serum activators could be nephritic factor, bacterial polysaccharides, polymeric IgA, activated properdin, peculiar immune complexes, or other factors. Such measurement is an improvement immunopathological step in the investigation of human GN

Changes of chondrocyte metabolism in vitro: an approach by proteomic analysis.

International audienceChanges in chondrocyte metabolism in vitro using different support systems and under different culture conditions were studied with a proteomic approach. Qualitative and quantitative modifications in the synthesis of chondrocyte proteins were investigated using two-dimensional (2D) gel electrophoresis. This technique provided a simple way to visualize the most abundant chondrocyte proteins. Proteins were identified after in-gel proteolysis with trypsin and matrix-assisted laser desorption ionization-time of flight mass spectrometry, using peptide mass fingerprinting. Tryptic peptide masses were measured and matched against a computer-generated list from the simulated trypsin proteolysis of a protein database (SwissProt).Changes in chondrocyte metabolism in vitro using different support systems and under different culture conditions were studied with a proteomic approach. Qualitative and quantitative modifications in the synthesis of chondrocyte proteins were investigated using two-dimensional (2D) gel electrophoresis. This technique provided a simple way to visualize the most abundant chondrocyte proteins. Proteins were identified after in-gel proteolysis with trypsin and matrix-assisted laser desorption ionization-time of flight mass spectrometry, using peptide mass fingerprinting. Tryptic peptide masses were measured and matched against a computer-generated list from the simulated trypsin proteolysis of a protein database (SwissProt)

Primers of contact system activity in asthmatic patients and contrast material reactors.

International audienceAn accelerated prekallikrein transformation is present at 4 degrees C in plasmas obtained from asthmatic patients (asthma plasmas) and in prereaction plasmas from contrast material (CM) reactors. Preliminary data show that (1) higher levels of alpha 2-macroglobulin bound kallikrein (alpha 2M-KK) may be found in asthma plasmas and pre-contrast medium reactor plasmas in comparison with control plasmas. In the asthma plasmas, these differences approach, but do not quite attain, significance; (2) complexes of alpha 2M-KK potentiate prekallikrein activation in buffer (BA) and dextran sulfate activation (DSA) assays; and (3) polybrene (factor XII inhibitor) induces an early blockage in the production of kallikrein activity in BA and DSA, indicating the presence in these plasmas of cryptic surfaces with a function analogous to dextran sulfate. These results indicate that, in asthma and probably in CM reactors plasmas, cryptic surfaces and alpha 2M-KK may each contribute to contact system activity induced at 4 degrees C.An accelerated prekallikrein transformation is present at 4 degrees C in plasmas obtained from asthmatic patients (asthma plasmas) and in prereaction plasmas from contrast material (CM) reactors. Preliminary data show that (1) higher levels of alpha 2-macroglobulin bound kallikrein (alpha 2M-KK) may be found in asthma plasmas and pre-contrast medium reactor plasmas in comparison with control plasmas. In the asthma plasmas, these differences approach, but do not quite attain, significance; (2) complexes of alpha 2M-KK potentiate prekallikrein activation in buffer (BA) and dextran sulfate activation (DSA) assays; and (3) polybrene (factor XII inhibitor) induces an early blockage in the production of kallikrein activity in BA and DSA, indicating the presence in these plasmas of cryptic surfaces with a function analogous to dextran sulfate. These results indicate that, in asthma and probably in CM reactors plasmas, cryptic surfaces and alpha 2M-KK may each contribute to contact system activity induced at 4 degrees C

[Anticomplementary activity of a polyanion: pentosan-poly-sulfoester, II.--Mode of action and "in vitro " inhibition of human complement hemolytic activity (author's transl)]

International audienceThe drug, pentosan-poly-sulfoester (PPS), is a potent in vitro inhibitor of the human complement hemolytic activity. This CH 50 inhibition represents a real anticomplementary activity (ACA), because this drug has no effect on sensitized sheep red blood cells (EA). The inhibition curve of human serum CH 50, by PPS is sigmoidal. The 50% inhibition is obtained for a 1: 650 dilution of PPS, which corresponds to a concentration of 0.08 mg/ml in normal human serum. Hemolytic titrations of C1, C4, C2, C3, and C5 showed a complete inhibition of C4, C2 and C3, and a partial inhibition of C1 (C1q, C1r, C1s, Ca++) and C5, by this drug. The mechanism of such functional inactivation of the different complement components is not yet elucidated.The drug, pentosan-poly-sulfoester (PPS), is a potent in vitro inhibitor of the human complement hemolytic activity. This CH 50 inhibition represents a real anticomplementary activity (ACA), because this drug has no effect on sensitized sheep red blood cells (EA). The inhibition curve of human serum CH 50, by PPS is sigmoidal. The 50% inhibition is obtained for a 1: 650 dilution of PPS, which corresponds to a concentration of 0.08 mg/ml in normal human serum. Hemolytic titrations of C1, C4, C2, C3, and C5 showed a complete inhibition of C4, C2 and C3, and a partial inhibition of C1 (C1q, C1r, C1s, Ca++) and C5, by this drug. The mechanism of such functional inactivation of the different complement components is not yet elucidated

[Anticomplement activity of a polyanion: pentosan sulfuric polyester. III. Mechanism of functional inactivation of the different properdin and complement system fractions]

International audienceIn vitro, the drug pentosan-poly-sulfoester (PPS) changes the electrophoretic migration of different proteins from the complement and properdin systems, such as native C3 (beta 1C globulin), C3c (beta-1A globulin), C3d (alpha-2D globulin), C1s inactivator (ClsINA), Clq and properdin factor B (B). Their more anodal migration is the consequence of a molecular alteration and persists after prolonged dialysis. These structural changes, yet undefined, explain the loss of functional activity of these proteins, and the anticomplementary activity of this drug. Moreover, PPS is able to block the alternate pathway activation by its action on properdin factor B (C3 proactivator). In fact, in presence of PPS, the activators of the properdin systems such as C3 nephritic factor are inactive. These altered mobilities are also responsible for the overestimation in the antigenic concentration of B (+ 45%), C4 (+27%) and C3/C3c (+ 14%), found in human serum containing 50 mg/ml of PPS. PPS has an original action upon the complement and properdin systems, which allows its clinical use as a potent inhibitor of the humoral mediators of inflammation.In vitro, the drug pentosan-poly-sulfoester (PPS) changes the electrophoretic migration of different proteins from the complement and properdin systems, such as native C3 (beta 1C globulin), C3c (beta-1A globulin), C3d (alpha-2D globulin), C1s inactivator (ClsINA), Clq and properdin factor B (B). Their more anodal migration is the consequence of a molecular alteration and persists after prolonged dialysis. These structural changes, yet undefined, explain the loss of functional activity of these proteins, and the anticomplementary activity of this drug. Moreover, PPS is able to block the alternate pathway activation by its action on properdin factor B (C3 proactivator). In fact, in presence of PPS, the activators of the properdin systems such as C3 nephritic factor are inactive. These altered mobilities are also responsible for the overestimation in the antigenic concentration of B (+ 45%), C4 (+27%) and C3/C3c (+ 14%), found in human serum containing 50 mg/ml of PPS. PPS has an original action upon the complement and properdin systems, which allows its clinical use as a potent inhibitor of the humoral mediators of inflammation

[Differentiation of adult human mesenchymal stem cells: Chondrogenic effect of BMP-2.]

International audienceArticular 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.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

Analogous features of cold-promoted activation and contact system activation in human plasmas: the role of cryptic soluble surfaces in asthma, oral contraception and pregnancy.

International audienceAspects of cold-promoted activation (CPA) and contact system activation (CSA) of human plasma were assayed in 13 normal subjects, 11 asthmatics and 15 patients with history of allergy to x-ray contrast media, to see whether the phenomena were correlated. Cold activation commonly occurs in stored plasma in 60% of women taking oral contraceptives, 93% of women in 3rd trimester of pregnancy, but only 15% of normal subjects. It is assayed by measuring kallikrein. CSA also occurs at low temperatures, but is dependent on soluble negatively-charged surfaces. It is assayed using dextran SO4 or polybrene. In this study, kallikrein amidolytic activity with dextran SO4-CDA and cold-dilution CDA were positively correlated in asthmatics. The allergic patients showing elevated B-CDA values also had shortened Thrombotest times with cold dilution, while those with normal B-CDA values had normal coagulation times. The significance of these results were discussed, including the theoretical possibility that cryptic surface phenomena may be involved in the heightened coagulability of plasma in women taking oral contraceptives.Aspects of cold-promoted activation (CPA) and contact system activation (CSA) of human plasma were assayed in 13 normal subjects, 11 asthmatics and 15 patients with history of allergy to x-ray contrast media, to see whether the phenomena were correlated. Cold activation commonly occurs in stored plasma in 60% of women taking oral contraceptives, 93% of women in 3rd trimester of pregnancy, but only 15% of normal subjects. It is assayed by measuring kallikrein. CSA also occurs at low temperatures, but is dependent on soluble negatively-charged surfaces. It is assayed using dextran SO4 or polybrene. In this study, kallikrein amidolytic activity with dextran SO4-CDA and cold-dilution CDA were positively correlated in asthmatics. The allergic patients showing elevated B-CDA values also had shortened Thrombotest times with cold dilution, while those with normal B-CDA values had normal coagulation times. The significance of these results were discussed, including the theoretical possibility that cryptic surface phenomena may be involved in the heightened coagulability of plasma in women taking oral contraceptives

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

International audienceIn 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).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)