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ABSTRACT: We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.
Tissue Engineering Part A 02/2009; 15(7):1523-32. · 4.64 Impact Factor
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ABSTRACT: CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.
Journal of Biological Chemistry 07/2008; 283(35):24254-63. · 4.77 Impact Factor
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ABSTRACT: Fetal bone cells were shown to have an interesting potential for therapeutic use in bone tissue engineering due to their rapid growth rate and their ability to differentiate into mature osteoblasts in vitro. We describe hereafter their capability to promote bone repair in vivo when combined with porous scaffolds based on poly(l-lactic acid) (PLA) obtained by supercritical gas foaming and reinforced with 5 wt.% beta-tricalcium phosphate (TCP). Bone regeneration was assessed by radiography and histology after implantation of PLA/TCP scaffolds alone, seeded with primary fetal bone cells, or coated with demineralized bone matrix. Craniotomy critical size defects and drill defects in the femoral condyle in rats were employed. In the cranial defects, polymer degradation and cortical bone regeneration were studied up to 12 months postoperatively. Complete bone ingrowth was observed after implantation of PLA/TCP constructs seeded with human fetal bone cells. Further tests were conducted in the trabecular neighborhood of femoral condyles, where scaffolds seeded with fetal bone cells also promoted bone repair. We present here a promising approach for bone tissue engineering using human primary fetal bone cells in combination with porous PLA/TCP structures. Fetal bone cells could be selected regarding osteogenic and immune-related properties, along with their rapid growth, ease of cell banking and associated safety.
Bone 04/2008; 42(3):554-64. · 4.02 Impact Factor
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ABSTRACT: Bioresorbable scaffolds made of poly(L-lactic acid) (PLA) obtained by supercritical gas foaming were recently described as suitable for tissue engineering, portraying biocompatibility with primary osteoblasts in vitro and interesting mechanical properties when reinforced with ceramics. The behavior of such constructs remained to be evaluated in vivo and therefore the present study was undertaken to compare different PLA/ceramic composite scaffolds obtained by supercritical gas foaming in a critical size defect craniotomy model in Sprague-Dawley rats. The host-tissue reaction to the implants was evaluated semiquantitatively and similar tendencies were noted for all graft substitutes: initially highly reactive but decreasing with time implanted. Complete bone-bridging was observed 18 weeks after implantation with PLA/ 5 wt % beta-TCP (PLA/TCP) and PLA/5 wt % HA (PLA/HA) scaffolds as assessed by histology and radiography. We show here for the first time that this solvent-free technique provides a promising approach in tissue engineering demonstrating both the biocompatibility and osteoconductivity of the processed structures in vivo.
Journal of Biomedical Materials Research Part A 11/2007; 83(1):41-51. · 2.63 Impact Factor
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ABSTRACT: Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.
The Journal of Immunology 07/2005; 174(11):6809-19. · 5.79 Impact Factor
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ABSTRACT: CD8+ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10-30-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an anti-parallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.
Journal of Biological Chemistry 06/2005; 280(25):23820-8. · 4.77 Impact Factor
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ABSTRACT: We envision the use of human fetal bone cells for engineered regeneration of adult skeletal tissue. A description of their cellular function is then necessary. To our knowledge, there is no description of human primary fetal bone cells treated with differentiation factors. The characterization of fetal bone cells is particularly important as the pattern of secreted proteins from osteoblasts has been shown to change during aging. In the first part of this work, human primary fetal bone cells were compared to adult bone cells and mesenchymal stem cells for their ability to proliferate and to differentiate into osteoblasts in vitro. Cell proliferation, gene expression of bone markers, alkaline phosphatase (ALP) activity, and mineralization were analyzed during a time-course study. In the second part of this paper, bone fetal cells behavior exposed to osteogenic factors is further detailed. The doubling time of fetal bone cells was comparable to mesenchymal stem cells but significantly shorter than for adult bone cells. Gene expression of cbfa-1, ALP, alpha1 chain of type I collagen, and osteocalcin were upregulated in fetal bone cells after 12 days of treatment, with higher inductions than for adult and mesenchymal stem cells. The increase of ALP enzymatic activity was stronger for fetal than for adult bone cells reaching a maximum at day 10, but lower than for mesenchymal stem cells. Importantly, the mineralization process of bone fetal cells started earlier than adult bone and mesenchymal stem cells. Proliferation of fetal and adult bone cells was increased by dexamethasone, whereas 1alpha,25-dihydroxyvitamin D3 did not show any proliferative effect. Mineralization studies clearly demonstrated the presence of calcium deposits in the extracellular matrix of fetal bone cells. Nodule formation and calcification were strongly increased by the differentiation treatment, especially by dexamethasone. This study shows for the first time that human primary fetal bone cells could be of great interest for bone research, due to their fast growth rate and their ability to differentiate into mature osteoblasts. They represent an interesting and promising potential for therapeutic use in bone tissue engineering.
Bone 01/2005; 35(6):1323-33. · 4.02 Impact Factor
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ABSTRACT: Fetal bone cells were shown to have an interesting potential for therapeutic use in bone tissue engineering due to their rapid growth rate and their ability to differentiate into mature ostcoblasts in vitro. We describe hereafter their capability to promote bone repair in vivo when combined with porous scaffolds based on poly(L-lactic acid) (PLA) obtained by supercritical gas foaming and reinforced with 5 wt.% beta-tricalcium phosphate (TCP).
