Full-Mouth Rehabilitation of a 15-Year-Old Girl Affected by a Rare Hypoparathyroidism (Glial Cell Missing Homolog 2 Mutation): A 3-Year Follow-Up.

OBJECTIVE: Familial isolated hypoparathyroidism is a rare genetic disorder due to no or low production of the parathyroid hormone, disturbing calcium and phosphate regulation. The resulting hypocalcemia may lead to dental abnormalities, such as enamel hypoplasia. The aim of this paper was to describe the full-mouth rehabilitation of a 15-year-old girl with chronic hypocalcemia due to a rare congenital hypoparathyroidism. CLINICAL CONSIDERATIONS: In this patient, in the young adult dentition, conservative care was preferred. Onlays or stainless-steel crowns were performed on the posterior teeth, and direct or indirect (overlays and veneerlays) were performed on the maxillary premolars, canines, and incisors, using a digital wax-up. The mandibular incisors were bleached. The treatment clearly improved the patient's oral quality of life, with fewer sensitivities, better chewing, and aesthetic satisfaction. The difficulties were the regular monitoring and the limited compliance of the patient. CONCLUSION: Despite no clinical feedback in the literature, generalized hypomineralized/hypoplastic teeth due to hypoparathyroidism in a young patient can be treated as amelogenesis imperfecta (generalized enamel defects) with a conservative approach for medium-term satisfactory results. HIGHLIGHTS: This study provides new insights into the management of enamel hypoplasia caused by familial isolated hypoparathyroidism, helping to improve patient outcomes in similar cases.

Efficient isolation of human gingival stem cells in a new serum-free medium supplemented with platelet lysate and growth hormone for osteogenic differentiation enhancement.

BACKGROUND: The use of distant autografts to restore maxillary bone defects is clinically challenging and has unpredictable outcomes. This variation may be explained by the embryonic origin of long bone donor sites, which are derived from mesoderm, whereas maxillary bones derive from neural crest. Gingival stem cells share the same embryonic origin as maxillary bones. Their stemness potential and ease of access have been repeatedly shown. One limitation in human cell therapy is the use of foetal calf serum during cell isolation and culture. To overcome this problem, a new serum-free medium enriched with an alternative to foetal calf serum, i.e., platelet lysate, needs to be adapted to clinical grade protocols. METHODS: Different serum-free media enriched with platelet lysate at various concentrations and supplemented with different growth factors were developed and compared to media containing foetal calf serum. Phenotypic markers, spontaneous DNA damage, and stem cell properties of gingival stem cells isolated in platelet lysate or in foetal calf serum were also compared, as were the immunomodulatory properties of the cells by co-culturing them with activated peripheral blood monocellular cells. T-cell proliferation and phenotype were also assessed by flow cytometry using cell proliferation dye and specific surface markers. Data were analysed with t-test for two-group comparisons, one-way ANOVA for multigroup comparisons and two-way ANOVA for repeated measures and multigroup comparisons. RESULTS: Serum-free medium enriched with 10% platelet lysate and growth hormone yielded the highest expansion rate. Gingival stem cell isolation and thawing under these conditions were successful, and no significant DNA lesions were detected. Phenotypic markers of mesenchymal stem cells and differentiation capacities were conserved. Gingival stem cells isolated in this new serum-free medium showed higher osteogenic differentiation potential compared to cells isolated in foetal calf serum. The proportion of regulatory T cells obtained by co-culturing gingival stem cells with activated peripheral blood monocellular cells was similar between the two types of media. CONCLUSIONS: This new serum-free medium is well suited for gingival stem cell isolation and proliferation, enhances osteogenic capacity and maintains immunomodulatory properties. It may allow the use of gingival stem cells in human cell therapy for bone regeneration in accordance with good manufacturing practice guidelines.

Involvement of neural crest and paraxial mesoderm in oral mucosal development and healing.

Tissue engineering therapies using adult stem cells derived from neural crest have sought accessible tissue sources of these cells because of their potential pluripotency. In this study, the gingiva and oral mucosa and their associated stem cells were investigated. Biopsies of these tissues produce neither scarring nor functional problems and are relatively painless, and fresh tissue can be obtained readily during different chairside dental procedures. However, the embryonic origin of these cells needs to be clarified, as does their evolution from the perinatal period to adulthood. In this study, the embryonic origin of gingival fibroblasts were determined, including gingival stem cells. To do this, transgenic mouse models were used to track neural crest derivatives as well as cells derived from paraxial mesoderm, spanning from embryogenesis to adulthood. These cells were compared with ones derived from abdominal dermis and facial dermis. Our results showed that gingival fibroblasts are derived from neural crest, and that paraxial mesoderm is involved in the vasculogenesis of oral tissues during development. Our in vitro studies revealed that the neuroectodermal origin of gingival fibroblasts (or gingival stem cells) endows them with multipotential properties as well as a specific migratory and contractile phenotype which may participate to the scar-free properties of the oral mucosa. Together, these results illustrate the high regenerative potential of neural crest-derived stem cells of the oral mucosa, including the gingiva, and strongly support their use in cell therapy to regenerate tissues with impaired healing.

[Oral hygiene prevention actions at Albert Chenevier hospital: Open day for schools]. / Actions de prévention bucco-dentaire à l’hôpital Albert Chenevier : journée « portes ouvertes ¼ pour les écoles.

BACKGROUND: Since 1994, the dental department of Albert Chenevier hospital in Créteil organizes an "open day" for schoolchildren in Creteil. This action is organized in collaboration with the Creteil Social Action Community Centre (CCAS) health prevention service and is part of a prevention programme designed to reduce caries prevalence in children aged 6 to 7 years. This programme helps especially children from disadvantaged groups or with disabilities. We want to report results for the last three years. METHODS: Allezard and Beauvin schools and La Nichée and Leloup special schools attended this open day, consisted of two parts. The first part concerned prevention, designed to teach children to brush their teeth. The second part consisted of screening of tooth decay. CCAS provided disposable toothbrushes, toothpaste and examination instruments. The Albert Chenevier hospital dental department provided hospital personnel, appropriate hygiene materials and premises. RESULTS: About 50% of children of all ages and from all schools presented tooth decay. The use of dental care decreased between 2011 and 2013 for Allezard and Beauvin schools. It was 100% in 2011 and 2012 for special schools and virtually zero for the Beauvin special school for disabled children. Dental care decreased from year to year for these special classes and for special schools. About 20% of children at the Allezard school had good oral hygiene. CONCLUSION: The effect of health education activities can be improved by involving the child's family. Oral hygiene must be based on a collective approach in which everyone has a role to play: children, parents, educators, teachers, principals and nursing staff should be involved to ensure continuing improvement of oral hygiene and to try to promote access to care. This preventive action therefore remains necessary and the information must be repeated.

Validation of Housekeeping Genes to Study Human Gingival Stem Cells and Their In Vitro Osteogenic Differentiation Using Real-Time RT-qPCR.

Gingival stem cells (GSCs) are recently isolated multipotent cells. Their osteogenic capacity has been validated in vitro and may be transferred to human cell therapy for maxillary large bone defects, as they share a neural crest cell origin with jaw bone cells. RT-qPCR is a widely used technique to study gene expression and may help us to follow osteoblast differentiation of GSCs. For accurate results, the choice of reliable housekeeping genes (HKGs) is crucial. The aim of this study was to select the most reliable HKGs for GSCs study and their osteogenic differentiation (dGSCs). The analysis was performed with ten selected HKGs using four algorithms: ΔCt comparative method, GeNorm, BestKeeper, and NormFinder. This study demonstrated that three HKGs, SDHA, ACTB, and B2M, were the most stable to study GSC, whereas TBP, SDHA, and ALAS1 were the most reliable to study dGSCs. The comparison to stem cells of mesenchymal origin (ASCs) showed that SDHA/HPRT1 were the most appropriate for ASCs study. The choice of suitable HKGs for GSCs is important as it gave access to an accurate analysis of osteogenic differentiation. It will allow further study of this interesting stem cells source for future human therapy.

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.

Endoluminal gingival fibroblast transfer reduces the size of rabbit carotid aneurisms via elastin repair.

OBJECTIVE: Matrix metalloproteinase-9 is considered to play a pivotal role in aneurismal formation. We showed that gingival fibroblasts (GF) in vitro reduced matrix metalloproteinase-9 activity via increased secretion of tissue inhibitor of metalloproteinase 1. We aimed to evaluate in vivo the efficacy of GF transplantation to reduce aneurism development in a rabbit model. METHODS AND RESULTS: Seventy rabbit carotid aneurisms were induced by elastase infusion. Four weeks later, GF, dermal fibroblast, or culture medium (DMEM) were infused into established aneurisms. Viable GF were abundantly detected in the transplanted arteries 3 months after seeding. GF engraftment resulted in a significant reduction of carotid aneurisms (decrease of 23.3% [P<0.001] and 17.6% [P=0.01] of vessel diameter in GF-treated arteries, 1 and 3 months after cell therapy, respectively), whereas vessel diameter of control DMEM and dermal fibroblast-treated arteries increased. GF inhibited matrix metalloproteinase-9 activity by tissue inhibitor of metalloproteinase 1 overexpression and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase 1 complex formation, induced elastin repair, and increased elastin density in the media compared with DMEM-treated arteries (38.2 versus 18.0%; P=0.02). Elastin network GF-induced repair was inhibited by tissue inhibitor of metalloproteinase 1 blocking peptide. CONCLUSIONS: Our results demonstrate that GF transplantation results in significant aneurism reduction and elastin repair. This strategy may be attractive because GF are accessible and remain viable within the grafted tissue.

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.

[Oropharyngeal candidiasis in elderly patients]. / Les candidoses oropharyngées des personnes âgées.

Oropharyngeal candidiasis is a common opportunistic infection of the oral cavity caused by an overgrowth of candida species, the commonest being Candida albicans. The prevalence in the hospital or institution varies from 13 to 47% of elderly persons. The main clinical types are denture stomatitis, acute atrophic glossitis, thrush and angular cheilitis. Diagnosis is usually made on clinical ground. Culture and sensitivity testing should be undertaken if initial therapy is unsuccessful. Predisposing factors of oral candidiasis could be local and/or systemic. Local factors include wearing dentures, impaired salivary gland function and poor oral health. Systemic factors include antibiotics and some other drugs, malnutrition, diabetes, immunosuppression and malignancies. Management involves an appropriate antifungal treatment and oral hygiene. Predisposing factors should be treated or eliminated where feasible. Oral hygiene involves cleaning the teeth and dentures. Dentures should be disinfected daily and left out overnight.

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.

Multipotent progenitor cells in gingival connective tissue.

The gum has an exceptional capacity for healing. To examine the basis for this property and explore the potential of conferring it to organs with inferior healing capacity, we sought the presence of progenitor cells in gingival connective tissue. Colony-forming units of fibroblast-enriched cells from gingival fibroblast cultures were assessed for expression of membrane markers of mesenchymal stem cells; capacity to differentiate into osteoblasts, chondroblasts, and adipocytes; and engraftment efficiency after in vivo transfer. On the basis of their ability to differentiate into several lineages, proliferate from single cells, induce calcium deposits, and secrete collagen in vivo after transfer on hydroxyapatite carriers, we suggest that this population represents gingival multipotent progenitor cells. The discovery of progenitor cells in gingival connective tissue may help improve our understanding of how the wounded gum is capable of almost perfect healing and opens the prospect of cellular therapy for wound healing using readily available cells at limited risk to the patient.

Fusiform aneurysm model in rabbit carotid artery.

AIMS: To develop a reproducible and accessible model of elastase-induced fusiform aneurysm in carotid rabbit arteries. METHODS: Elastase, at a concentration of 1-30 U, was incubated into the lumen of carotid rabbit arteries. Four weeks later, angiography, histomorphometry, immunohistochemistry and zymography were performed. RESULTS: The optimal concentration of elastase in this model was 3 U according to the balance between mortality and thrombosis rates. Indeed, at 3 U, external carotid diameter increased from 1.9 +/- 0.1 to 3.1 +/- 0.4 mm (p < 0.0001) associated with degradation of elastic fibers, matrix metalloproteinase-9 secretion, apoptosis and macrophage infiltration. CONCLUSIONS: Our study underlines that abdominal aortic aneurysm can be reliably duplicated in an elastase-induced aneurysm in carotid artery, a much more accessible vessel.

Gingival fibroblast inhibits MMP-7: evaluation in an ex vivo aorta model.

Matrix metalloproteinases (MMP) play a deleterious role in numerous vascular diseases. In contrast, gingival matrix remodelling is adequately regulated by the gingival fibroblast (GF). Here, we aimed to evaluate the GF activity on MMP-7 expression and secretion in coculture with aorta rings. We evaluated MMP-7 transcription and secretion in rabbit aorta rings cultured or not with gingival fibroblasts in collagen gels. GF induced an increase of TIMP-1 transcription and secretion, followed, similarly to other MMPs, by the formation of TIMP-1/MMP-7 complexes. There was also a decrease of MMP-7 mRNA by RT-PCR in aorta rings cocultured with gingival fibroblasts. Interestingly, in contrast with other MMPs (which were not influenced at a transcription level), GF stimulated the release of TGF-beta1, which in turn inhibited the transcription and synthesis of MMP-7, as shown by neutralizing MMP-7 inhibition due to gingival fibroblast by overexpressing decorin (a TGF beta 1 inhibitor) or by silencing TGF beta 1 using siRNA. We showed that healing properties of the GF could be transposed to another organ, i.e., ex vivo aneurism model, implicating a down-regulation of MMP-7.

Gingival fibroblasts inhibit MMP-1 and MMP-3 activities in an ex-vivo artery model.

The main arterial pathologies can be associated with a deregulation of remodeling involving matrix metalloproteinases (MMPs), whereas gingival healing is characterized by an absence of fibrosis or irreversible elastin/collagen degradation. The aim of our study was to evaluate the effect of gingival fibroblasts on MMP-1 and MMP-3 secretion in an organotypic artery culture. MMP-1 and MMP-3 secretions and activities (dot blots, zymography, ELISA) were evaluated in coculture of rabbit artery in the presence or not of gingival fibroblasts. MMP-1/TIMP-1 and MMP-3/TIMP-1 complexes forms were measured by ELISA. Complementary studies were performed using human aortic smooth muscle cells cocultured with adventitial, dermal, or gingival fibroblasts. Our results indicated that MMP-1 and MMP-3 free-forms activities were significantly reduced in coculture. This inhibition was linked to a significant increase of TIMP-1 leading to formation of TIMP-1/MMPs complexes. Due to the presence of gingival fibroblasts, the decrease in MMP-1 and MMP-3 efficiency thus contributes to diminish the degradation of artery. This cellular therapy strategy could be promising in artery pathologies treatment.

Preservation of rabbit aorta elastin from degradation by gingival fibroblasts in an ex vivo model.

OBJECTIVE: Embryo-like gingival healing properties are attributed to the gingival fibroblast (GF) and could be used as a model for other types of healing dysfunctions. Abdominal aortic aneurysm (AAA) formation is associated with elastin degradation and increase in matrix metalloproteinase (MMP)-9 activity. We aimed to validate the concept of using GF healing properties in arteries. METHODS AND RESULTS: We evaluated MMP-9 and its tissue inhibitor (TIMP-1) in rabbit aortic rings cultured in collagen gels with or without GFs and observed throughout 21 days. We also performed cocultures of human smooth muscle cells (hSMCs) with either gingival, dermal, or adventitial fibroblasts, and alone (control). In control arteries, elastic fibers became spontaneously sparse. In presence of GFs, elastic fibers were preserved. There was a dramatically reduced protein level of MMP-9 in coculture of aorta and GFs, in contrast with control aorta. MMP-9 expression was unaffected by GFs. MMP-9 inhibition was related to increased TIMP-1 secretion, TIMP-1 forming a complex with MMP-9. Cell cocultures of hSMC with GFs showed similar results. Dermal and adventitial fibroblasts did not affect MMP-9. CONCLUSIONS: Elastic fiber degradation was specifically preserved by GFs via reduction of MMP-9 protein level by increasing TIMP-1 synthesis. Vascular transfer of gingival fibroblasts could be a promising approach to treat AAA.

Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution.

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates geared to simplified preparation without biochemical blood handling. In this initial article, we describe the conceptual and technical evolution from fibrin glues to platelet concentrates. This retrospective analysis is necessary for the understanding of fibrin technologies and the evaluation of the biochemical properties of 3 generations of surgical additives, respectively fibrin adhesives, concentrated platelet-rich plasma (cPRP) and PRF. Indeed, the 3-dimensional fibrin architecture is deeply dependent on artificial clinical polymerization processes, such as massive bovine thrombin addition. Currently, the slow polymerization during PRF preparation seems to generate a fibrin network very similar to the natural one. Such a network leads to a more efficient cell migration and proliferation and thus cicatrization.

Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates, with simplified processing and without biochemical blood handling. In this second article, we investigate the platelet-associated features of this biomaterial. During PRF processing by centrifugation, platelets are activated and their massive degranulation implies a very significant cytokine release. Concentrated platelet-rich plasma platelet cytokines have already been quantified in many technologic configurations. To carry out a comparative study, we therefore undertook to quantify PDGF-BB, TGFbeta-1, and IGF-I within PPP (platelet-poor plasma) supernatant and PRF clot exudate serum. These initial analyses revealed that slow fibrin polymerization during PRF processing leads to the intrinsic incorporation of platelet cytokines and glycanic chains in the fibrin meshes. This result would imply that PRF, unlike the other platelet concentrates, would be able to progressively release cytokines during fibrin matrix remodeling; such a mechanism might explain the clinically observed healing properties of PRF.