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ABSTRACT: 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.
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.
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.
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.
Osteoarthritis and Cartilage 08/2006; 14(7):631-40. · 3.90 Impact Factor
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ABSTRACT: To determine the effects of osteoarthritic (OA) subchondral osteoblasts on the metabolism of human OA chondrocytes in alginate beads.
Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 days in the absence or in the presence of osteoblasts isolated from non-sclerotic (N) or sclerotic (SC) zones of human OA subchondral bone in monolayer (co-culture system). Before co-culture, osteoblasts were incubated for 72 h with or without 1.7ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M (OSM). Aggrecan (AGG) and matrix metalloproteases (MMP)-3 and -13 mRNA levels in chondrocytes were quantified by real-time polymerase chain reaction. AGG production was assayed by a specific enzyme amplified sensitivity immunoassay.
SC, but not N, osteoblasts induced a significant inhibition of AGG production and AGG gene expression by human OA chondrocytes in alginate beads, and significantly increased MMP-3 and MMP-13 gene expression by chondrocytes. When they were pre-incubated with IL-1beta, IL-6 or OSM, N osteoblasts inhibited AGG synthesis and increased MMP-3 and -13 gene expression by chondrocytes in alginate beads in a same order of magnitude as SC osteoblasts.
These results demonstrate that SC OA subchondral osteoblasts could contribute to cartilage degradation by stimulating chondrocytes to produce more MMP and also by inhibiting AGG synthesis.
Osteoarthritis and Cartilage 12/2005; 13(11):979-87. · 3.90 Impact Factor
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ABSTRACT: To determine the influence of osteoarthritic (OA) phenotype of subchondral osteoblasts on the phenotype of human chondrocytes.
Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 or 10 days in the absence or in the presence of osteoblasts in monolayer. The osteoblasts were either isolated from non-sclerotic (N) or sclerotic (SC) zones of human subchondral bone. Before co-culture, osteoblasts were incubated for 72 h with or without 1.7 ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M. SOX9, type I, II and X collagen (COL1, COL2, COL10), osteoblasts-stimulating factor (OSF)-1, bone alkaline phosphatase (ALP), parathyroid hormone related peptide (PTHrP) and its receptor (PTH-R) messenger RNA (mRNA) levels in chondrocytes were quantified by real-time polymerase chain reaction.
In comparison with chondrocytes cultured alone in alginate beads, chondrocytes after 4 days in co-culture with N or SC osteoblasts expressed significantly less SOX9 and COL2 mRNA. The decrease of SOX9 and COL2 gene expression was significantly more pronounced in the presence of SC than in the presence of N osteoblasts (P<0.001). OSF-1 mRNA level in chondrocyte was increased by both N and SC osteoblasts, but to a larger extent by SC osteoblasts (P<0.001). PTHrP expression in chondrocytes was 21-fold increased by N osteoblasts but four-fold inhibited by SC osteoblasts. PTHrP secretion was also increased by N but reduced by SC osteoblasts. SC, but not N osteoblasts, induced a significant decrease of PTH-R gene expression in chondrocyte. In our experimental conditions, chondrocytes did not express COL1, COL10 or ALP, even after 10 days of co-culture with osteoblasts.
In co-culture, SC subchondral osteoblasts decrease SOX9, COL2, PTHrP and PTH-R gene expression by chondrocytes but increase that of OSF-1. These findings suggest that SC osteoblasts could initiate chondrocyte phenotype shift towards hypertrophic differentiation and subsequently further matrix mineralization.
Osteoarthritis and Cartilage 11/2005; 13(11):988-97. · 3.90 Impact Factor
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ABSTRACT: Migration and maturation of epidermal dendritic cells, the Langerhans cells (LC), are central events in the initiation of the cutaneous immune response. LC migration from skin to draining lymph nodes is regarded as an indispensable step for the early phase of antigen-specific sensitization. Among the several agents which influence the ability of LC to migrate, previous studies have revealed that matrix metalloproteinases (MMPs) and protein kinase C (PKC) contribute to promoting LC migration. In this work, we studied the effect of two recently developed PKC and MMPs inhibitors of vegetable origin on the migration of in vitro activated LC.
The migratory capacity of epidermal and in vitro generated LC was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to the lymph nodes.
Contact with chemical allergens, Bandrowski's base or 2,4-dinitrobenzenesulfonic acid (DNBS), triggered migration. In the presence of PKC inhibitors, D-erythro-sphingosine and OX100, or an inhibitor of MMPs, LU105, allergen-induced migration of LC was strongly decreased. The association between OX100 and LU105 was more efficient in modulating the migration of activated LC compared to each molecule tested separately.
These results showed that PKC and MMPs inhibitors act in synergy to inhibit the migration of activated epidermal dendritic cells in vitro. They underscore the role of PKC and MMPs inhibitors and suggest they may be of relevance for therapeutically regulating epidermal dendritic cell migration in inflammatory dermatoses.
International Archives of Allergy and Immunology 05/2004; 133(4):348-56. · 2.40 Impact Factor
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ABSTRACT: This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture. LU105 was extracted from seeds of L. albus and is freely soluble in water. Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48 h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected. Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B). LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9. Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue. TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts. Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva.
Clinical Oral Investigations 01/2004; 7(4):198-205. · 2.36 Impact Factor
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ABSTRACT: The aim of this study was to determine if a vegetable extract from seeds of Lupinus albus (LU 105) has the capacity to inhibit human leukocyte elastase and/or protect gingival elastic fibers against proteolytic degradation. LU105 was extracted from seeds of L albus and is freely soluble in water. In this study the ex-vivo elastolytic activity of human leukocyte elastase and the potential inhibitory effect of LU 105 were determined using human gingival cryostat tissue sections and computerized morphometric analysis. The gingival tissue sections pre-treated or not with LU 105 were submitted to the action of human leukocyte elastase or submitted to the simultaneous action of human leukocyte elastase and LU 105, and then analyzed using automated image analysis. In such conditions, LU 105 at 0.1%, 0.01%, and 0.001% developed a dose dependent protection of gingival elastic fibers against enzymatic proteolysis due to human leukocyte elastase, and LU 105 at 0.1% or 0.01% was able to inhibit the elastolytic activity of leukocyte elastase itself. It is proposed that LU 105 is an option for the treatment of gingival inflammation in which leukocyte elastase is involved.
Clinical Oral Investigations 01/2004; 7(4):206-11. · 2.36 Impact Factor
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ABSTRACT: The aim of this study was to determine if a vegetable extract from seeds of Lupinus albus (LU 105) has the capacity to inhibit human leukocyte elastase and/or protect gingival elastic fibers against proteolytic degradation. LU105 was extracted from seeds of L albus and is freely soluble in water. In this study the ex-vivo elastolytic activity of human leukocyte elastase and the potential inhibitory effect of LU 105 were determined using human gingival cryostat tissue sections and computerized morphometric analysis. The gingival tissue sections pre-treated or not with LU 105 were submitted to the action of human leukocyte elastase or submitted to the simultaneous action of human leukocyte elastase and LU 105, and then analyzed using automated image analysis. In such conditions, LU 105at 0.1%, 0.01%, and 0.001% developed a dose dependent protection of gingival elastic fibers against enzymatic proteolysis due to human leukocyte elastase, and LU 105at 0.1% or 0.01% was able to inhibit the elastolytic activity of leukocyte elastase itself. It is proposed that LU 105 is an option for the treatment of gingival inflammation in which leukocyte elastase is involved.
Clinical Oral Investigations 11/2003; 7(4):206-211. · 2.36 Impact Factor
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ABSTRACT: This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture. LU105 was extracted from seeds of L. albus and is freely soluble in water. Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected. Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B). LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9. Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue. TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts. Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva.
Clinical Oral Investigations 01/2003; 7(4):198-205. · 2.36 Impact Factor
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ABSTRACT: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts.
Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis.
In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml.
These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.
Journal of Periodontology 01/2002; 72(12):1685-94. · 2.60 Impact Factor
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Annales de Dermatologie et de Vénéréologie 129(8-9):1071-7. · 0.72 Impact Factor
