Phenotypic Study of Human Gingival Fibroblasts in a Medium Enriched With Platelet Lysate
Institute for Health and Medical Research - Unit 849, Paris Descartes University, Health Services-Paris Hospitals, Paris, France. Journal of Periodontology
(Impact Factor: 2.71).
11/2010; 82(4):632-41. DOI: 10.1902/jop.2010.100179
The modulation abilities of gingival fibroblasts open new therapeutic strategies for the treatment of vascular diseases (e.g., aneurism) and irradiation burns. Culture media are classically supplemented with animal sera to provide nutriments. Unfortunately, because of their potential for interspecies transmission of microorganisms, these media are not used for cells destined for human transplantation. This preliminary phenotypic study aims to test a serum-free (SF) culture medium for human gingival fibroblasts (hGF) supplemented with human platelet lysates (PLs) for rapid cell expansion.
An SF medium was first elaborated to compete with hGF proliferation in a reference medium containing 10% fetal bovine serum (BSmedium). Adhesion, proliferation, and doubling kinetics were run in the presence of PLs (SF+PL). Cytoskeletal proteins were analyzed and chromosomal abnormalities were evaluated by karyotype analyses. The SF+PL influence on secretion of molecules implied in tissue remodeling (i.e., matrix metalloproteinases [MMPs], their tissue inhibitors [TIMPs], and several growth factors) was studied.
SF+PL increased the proliferation rate 1.5-fold in a week compared to BSmedium. Cytoskeleton protein expression was similar in BSmedium and in SF+PL. Chromosomal abnormalities were rare in SF+PL. MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, TIMP-1, and the growth factors interleukin-1β and -4 and transforming growth factor-β1 secretions were stable during the experiment. TIMP-2 and interleukin-6 were slightly decreased in SF+PL compared to BSmedium.
While waiting confirmation from a proteomic approach, this SF culture medium could allow a secured faster hGF proliferation adapted for human cell transplant therapy.
Available from: François Côme Ferré
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ABSTRACT: Skin aging shows an imbalance between synthesis and degradation of the extracellular matrix. The overproduction of degradative enzymes (MMPs) during the chronology-and photo-induced aging leads to a degradation of the elastic and collagen networks. In a model of collagen and elastin destruction, we showed that the gingival fibroblast was able to preserve these macromolecules by inhibiting the overproduction of metalloproteinases by overproduction of TIMP-1 and modulation of the inflammatory cytokines activity. The objective of this study is to evaluate the effect of the gingival fibroblasts on human skin. The results in vitro and ex vivo show that the gingival fibroblast protects the skin collagen and elastic network by the inhibition of MMPs which leads to an overproduction of the TIMP-1. Moreover, the gingival fibroblast modulates the activity of some enzymes responsible for the inflammation; they inhibit the IL-1β and stimulate the production of TGF-β1. In vivo studies with a duration of six months and 50 women with pronounced wrinkles show that the culture supernatant of gingival fibroblasts diluted to 5% leads to a statistically significant decrease in the number and length of wrinkles.
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ABSTRACT: Introduction: Dental pulp stem cells (DPSCs) are an accessible cell source with therapeutic applicability in
regeneration of damaged tissues. Current techniques for expansion of DPSCs require the use of Fetal Bovine Serum
(FBS). However, animal-derived reagents stage safety issues in clinical therapy. By expanding DPSCs in serumfree/
xenofree medium (SF/XF-M) or in medium containing human serum (HS-M), the problems can be eliminated.
Therefore, the aim of our study was to identify suitable cell culture media alternatives for DPSCs.
Methods: We studied the isolation, proliferation, morphology, cell surface markers (CD29, CD44, CD90,
CD105, CD31, CD45 and CD146), stemness markers expression (Oct3/4, Sox2, Nanog and SSEA-4) and in vitro
multilineage differentiation of DPSCs in HS-M or SF/XF-M in comparison to FBS-M.
Results: DPSCs expressed the cell surface and stemness markers in all studied conditions. The proliferation
analysis of cells cultured in different HS concentrations revealed that cells isolated in 20% HS-M and passaged in
10% or 15% HS-M supported the cell growth. Direct isolation of cells in SF/XF-M did not support cell proliferation.
Therefore, cells cultured in 20% HS-M were used for further SF/XF-M studies. However, proliferation of DPSCs
was significantly lower in SF/XF-M when compared with cells cultured in FBS-M and HS-M. In addition, proliferation
of DPSCs in SF/XF-M could be enhanced by addition of 1% HS in cell culture medium. There were differences in
osteogenic, chondrogenic and adipogenic differentiation efficacy between cells cultured in FBS, HS and SF/XF
differentation media. More pronounced adipogenic and osteogenic differentiation was observed in HS differentiation
medium, however, in FBS-M cultured cells more effective chondrogenic differentiation was detected.
Conclusions: Our results indicate that HS is a suitable alternative to FBS for the expansion of DPSCs. The
composition of SF/XF-M needs to be further optimized in terms of cell expandability and differentiation efficiency to
reach clinical applicability.
Available from: onlinelibrary.wiley.com
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ABSTRACT: Platelet gels (PG) are blood-derived biomaterials that are generally obtained through the activation of a platelet-rich plasma or a platelet concentrate by thrombin or calcium chloride, resulting in the simultaneous conversion of fibrinogen into a fibrin gel and in the generation of a platelet releasate rich in a physiological cocktail of growth factors. To reinforce the physical strength of the fibrin network, a fibrinogen-rich fraction – generally cryoprecipitate – can be added to the platelet fraction prior to activation, resulting in the generation of platelet fibrin glue (PFG). PG and PFG, prepared from single donations, either autologous or allogeneic, are increasingly used, alone or in combination with grafting materials, in various field of regenerative medicine where the presence of growth factors is expected to stimulate the healing of soft or hard tissues. Being obtained from human blood, they are physiological and biodegradable preparations and do not induce tissue necrosis. So far, the viral safety of most allogeneic PG and PFG relies on donors selection and donation testing, as is the case for all non-virally inactivated blood components for transfusion. Major fields of clinical applications of PG and PFG in osseous tissue regeneration include maxillo-facial surgery, implantology, reconstructive and plastic surgery. Platelet gels are also used for enhancing the healing of soft tissues, most particularly recalcitrant lower extremity ulcers of various aetiologies, and burns. Newer promising indications include the treatment of osteo-arthritis and joint inflammation, and the repair of musculoskeletal tissue lesions in sports medicine. Autologous PG and PFG are mostly ‘home-made’ single-donor preparations prepared using medical devices. They suffer from the variability in donor characteristics and in isolation procedures of the platelet fraction. Clinical application methods are not standardized. Variability in autologous product characteristics is high, and optimal content of growth factors is unknown, confusing the analysis of product efficacy. The evidence of clinical benefits of these products based on controlled clinical studies is lacking in most indications, although many case studies do support an objective benefit in soft and probably hard tissues healing. Improvement in the standardization and formulation of PG and PFG is a mandatory step forward for improving the reliability and the predictability of clinical outcomes of these interesting blood preparations.
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