Extracellular Matrix Derived From Dental Pulp Stem Cells Promotes Mineralization.

Background: Extracellular matrix (ECM) plays a pivotal role in many physiological processes. ECM macromolecules and associated factors differ according to tissues, impact cell differentiation, and tissue homeostasis. Dental pulp ECM may differ from other oral tissues and impact mineralization. Thus, the present study aimed to identify the matrisome of ECM proteins derived from human dental pulp stem cells (DPSCs) and its ability to regulate mineralization even in cells which do not respond to assaults by mineralization, the human gingival fibroblasts (GF). Methods: ECM were extracted from DPSCs cultured in normal growth medium supplemented with L-ascorbic acid (N-ECM) or in osteogenic induction medium (OM-ECM). ECM decellularization (dECM) was performed using 0.5% triton X-100 in 20 mM ammonium hydroxide after 21 days. Mass spectrometry and proteomic analysis identified and quantified matrisome proteins. Results: The dECM contained ECM proteins but lacked cellular components and mineralization. Interestingly, collagens (COL6A1, COL6A2, and COL6A3) and elastic fibers (FBN1, FBLN2, FN1, and HSPG2) were significantly represented in N-ECM, while annexins (ANXA1, ANXA4, ANXA5, ANXA6, ANXA7, and ANXA11) were significantly overdetected in OM-ECM. GF were reseeded on N-dECM and OM-dECM and cultured in normal or osteogenic medium. GF were able to attach and proliferate on N-dECM and OM-dECM. Both dECM enhanced mineralization of GF at day 14 compared to tissue culture plate (TCP). In addition, OM-dECM promoted higher mineralization of GF than N-dECM although cultured in growth medium. Conclusions: ECM derived from DPSCs proved to be osteoinductive, and this knowledge supported cell-derived ECM can be further utilized for tissue engineering of mineralized tissues.

Comparative study of abdominal and thoracic aortic aneurysms: their pathogenesis and a gingival fibroblasts-based ex vivo treatment.

Aortic aneurysms (AAs) consist of slow proteolysis and loss of both collagen and elastin matrix in the aorta wall, leading to wall dilation, weakening and rupture in well-advanced lesions. This can occur in both abdominal aorta (Abdominal Aortic Aneurysm: AAA) and thoracic aorta (Thoracic Aortic Aneurysm: TAA). To date, no non-surgical therapy has been proposed to slow or stop AA progression. Previously published preclinical studies from our team using an aneurysm rabbit model showed a promising concept for treatment of AAs with gingival fibroblast (GFs) which are readily available cells. In this study, we investigated the possible tissue repair of human AAAs and TAAs using ex vivo models co-cultured with GFs. Histological analysis showed that TAA and AAA are two distinct pathologies. Both lesions presented destruction of the aorta wall, highly evidenced in AAA samples. The results have confirmed the presence of the bacterial Porphyromonas gingivalis (Pg) protein in all AAA samples, but not in TAA samples, indicating the possible role of an infectious factor in the developing and progression of AAA lesions compared to TAA. The co-culture of GFs with AA lesions shows increased expression of TIMP-1, the inhibitor of the aneurysm severity marker MMP-9. Our study indicates that GFs might ameliorate aorta wall reestablishment in both AA types by their regenerative and immunomodulatory capacities. It also demonstrates the possible infectious cause of AAA compared with TAA that may explain their different behavior.

Formation of cartilage and synovial tissue by human gingival stem cells.

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis.

Oral phenotype and scoring of vascular Ehlers-Danlos syndrome: a case-control study.

OBJECTIVE: Vascular Ehlers-Danlos syndrome (vEDS) is a rare genetic condition related to mutations in the COL3A1 gene, responsible of vascular, digestive and uterine accidents. Difficulty of clinical diagnosis has led to the design of diagnostic criteria, summarised in the Villefranche classification. The goal was to assess oral features of vEDS. Gingival recession is the only oral sign recognised as a minor diagnostic criterion. The authors aimed to check this assumption since bibliographical search related to gingival recession in vEDS proved scarce. DESIGN: Prospective case-control study. SETTING: Dental surgery department in a French tertiary hospital. PARTICIPANTS: 17 consecutive patients with genetically proven vEDS, aged 19-55 years, were compared with 46 age- and sex-matched controls. OBSERVATIONS: Complete oral examination (clinical and radiological) with standardised assessment of periodontal structure, temporomandibular joint function and dental characteristics were performed. COL3A1 mutations were identified by direct sequencing of genomic or complementary DNA. RESULTS: Prevalence of gingival recession was low among patients with vEDS, as for periodontitis. Conversely, patients showed marked gingival fragility, temporomandibular disorders, dentin formation defects, molar root fusion and increased root length. After logistic regression, three variables remained significantly associated to vEDS. These variables were integrated in a diagnostic oral score with 87.5% and 97% sensitivity and specificity, respectively. CONCLUSIONS: Gingival recession is an inappropriate diagnostic criterion for vEDS. Several new specific oral signs of the disease were identified, whose combination may be of greater value in diagnosing vEDS.

Phenotypic study of human gingival fibroblasts in a medium enriched with platelet lysate.

BACKGROUND: 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. METHODS: 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. RESULTS: 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-1ß and -4 and transforming growth factor-ß1 secretions were stable during the experiment. TIMP-2 and interleukin-6 were slightly decreased in SF+PL compared to BSmedium. CONCLUSION: While waiting confirmation from a proteomic approach, this SF culture medium could allow a secured faster hGF proliferation adapted for human cell transplant therapy.