Human dental pulp stem cells (hDPSCs): isolation, enrichment and comparative differentiation of two sub-populations.

BACKGROUND: Human dental pulp represents a suitable alternative source of stem cells for the purpose of cell-based therapies in regenerative medicine, because it is relatively easy to obtain it, using low invasive procedures. This study characterized and compared two subpopulations of adult stem cells derived from human dental pulp (hDPSCs). Human DPSCs, formerly immune-selected for STRO-1 and c-Kit, were separated for negativity and positivity to CD34 expression respectively, and evaluated for cell proliferation, stemness maintenance, cell senescence and multipotency. RESULTS: The STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs showed a slower proliferation, gradual loss of stemness, early cell senescence and apoptosis, compared to STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs. Both the subpopulations demonstrated similar abilities to differentiate towards mesoderm lineages, whereas a significant difference was observed after the neurogenic induction, with a greater commitment of STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs. Moreover, undifferentiated STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs did not show any expression of CD271 and nestin, typical neural markers, while STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs expressed both. CONCLUSIONS: These results suggest that STRO-1(+)/c-Kit(+)/CD34(-) hDPSCs and STRO-1(+)/c-Kit(+)/CD34(+) hDPSCs might represent two distinct stem cell populations, with different properties. These results trigger further analyses to deeply investigate the hypothesis that more than a single stem cell population resides within the dental pulp, to better define the flexibility of application of hDPSCs in regenerative medicine.

Oral prevalence and clearance of oncogenic human papilloma virus in a rehabilitation community for substance abusers in Italy: a case of behavioral correction?

BACKGROUND: Human papilloma virus oral infection can be related to several factors including HIV infection, cigarette smoking, marijuana consumption and number of sexual partners. We conducted a study on oral HPV prevalence and clearance among the hosts of the San Patrignano community, a population considered at "high-risk" for HPV due to their previous habits. METHODS: From March 2007 to September 2010 all subjects were submitted to oropharyngeal brushing and saliva collection at baseline, after 6, 12 and 48 months (for subjects HPV positive at baseline). Samples were analyzed to detect HPV DNA and virus genotypes. The correlation between HPV prevalence and demographic, behavioral or immunological characteristics was assessed. RESULTS: Among 194 subjects, 30 (15%) were HPV positive with 25 (13%) high-risk (HR)-HPV at baseline brushing. At 12 months HPV infection was cleared in all cases. However at 48 months HPV was newly detected in 33% of subjects. A correlation between time spent in the community and increase in the ratio of "low-risk" (LR) HPV and HR-HPV was observed. HPV infection was not associated with age, gender, HIV status, HCV, alcohol and/or drug exposure, number of years spent in community, sex with drug-addicts and condom use. Only AIDS under antiretroviral treatment was inversely correlated with the risk of infection. CONCLUSIONS: At 1 year a complete HPV clearance was observed which could be related to adoption of healthier lifestyles of participants. New HPV infections were detected even in the absence of the recognized and declared risky behavioral factors, suggesting a re-expression from a latent infection.

Variations in inflammatory genes are associated with periodontitis.

BACKGROUND: Periodontitis is a multi-factorial disease and several risk-factors such as infections, inflammatory responses, oral hygiene, smoke, aging and individual predisposition are involved in the disease. Pathogens trigger chronic inflammation with cytokines release which in turn leads to the destruction of the connective and the teeth supporting bone. The identification of genetic factors controlling oral inflammation may increase our understanding of genetic predisposition to periodontitis.Single nucleotide polymorphisms in the promoter region of Vascular Endothelial Growth Factor, Alpha-1-Antichymotripsin, hydroxy-methyl-glutaryl CoA reductase, Interferon alpha, Interleukin-1 Beta, Interleukin 10, Interleukin 6 and Tumor Necrosis Factor- alpha genes from a case/control study were investigated. RESULTS: The C allele of Vascular Endothelial Growth Factor, A allele of Interleukin 10 and GG genotype of Tumor Necrosis Factor-α were individually associated with chronic periodontitis. However, the concomitant presence of the three genetic markers in the same subjects appeared to play a synergistic role and increased several folds the risk of the disease. CONCLUSIONS: Our findings offer new tools to implement the screening of unaffected subjects with an increased susceptibility of periodontitis and increase our understanding regarding the genetic inflammatory background related to familiarity of the disease.

Phytochemical composition and in vitro screening of the antimicrobial activity of essential oils on oral pathogenic bacteria.

In this study, the activity of essential oils (EOs) against microorganisms involved in oral diseases was evaluated. Fourteen EOs were selected and subjected to gas chromatographic analysis, including Illicium verum, Eucaliptus globulus, Eugenia caryophyllata, Leptospermum scoparium, Mentha arvensis, Mentha piperita, Myrtus communis, Salvia officinalis, Melaleuca alternifolia, Rosmarinus officinalis, Lavandula x intermedia, Thymus capitatus and Thymus vulgaris. These EOs were tested for their antimicrobial activity on Streptococcus mutans and Lactobacillus species clinically isolated from dental surgery patients. The antibacterial activity was evaluated by means of the disc diffusion and the minimum inhibitory concentration (MIC). Five EOs, having shown an interesting antimicrobial activity, were selected for a second screening in combination between them and with chlorhexidine. From the second assays, two EO-EO and three EO-chlorhexidine associations gave interesting results as potential constituents of mouthwashes, especially for the contribution of oxygenated monoterpenes, including menthol, thymol and carvacrol.

Human dental pulp stem cells expressing STRO-1, c-kit and CD34 markers in peripheral nerve regeneration.

Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The application of stem cells able to differentiate in Schwann cell-like cells in vitro and in vivo, could represent an attractive therapeutic approach for the treatment of nerve injuries. Further, stem cells sources sharing the same embryological origin as Schwann cells might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal subpopulation of human STRO-1+ /c-Kit+ /CD34+ DPSCs, expressing P75NTR , nestin and SOX-10, to differentiate into Schwann cell-like cells in vitro and to promote axonal regeneration in vivo, which led to functional recovery as measured by sustained gait improvement, in animal rat model of peripheral nerve injury. Transplanted human dental pulp stem cells (hDPSCs) engrafted into sciatic nerve defect, as revealed by the positive staining against human nuclei, showed the expression of typical Schwann cells markers, S100b and, noteworthy, a significant number of myelinated axons was detected. Moreover, hDPSCs promoted axonal regeneration from proximal to distal stumps 1 month after transplantation. This study demonstrates that STRO-1+ /c-Kit+ /CD34+ hDPSCs, associated with neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues. Copyright © 2016 John Wiley & Sons, Ltd.

Optimized Cryopreservation and Banking of Human Bone-Marrow Fragments and Stem Cells.

Adult mesenchymal stem cells are a promising source for cell therapies and tissue engineering applications. Current procedures for banking of human bone-marrow mesenchymal stem cells (hBM-MSCs) require cell isolation and expansion, and thus the use of large amounts of animal sera. However, animal-derived culture supplements have the potential to trigger infections and severe immune reactions. The aim of this study was to investigate an optimized method for cryopreservation of human bone-marrow fragments for application in cell banking procedures where stem-cell expansion and use are not immediately needed. Whole trabecular fragments enclosing the bone marrow were stored in liquid nitrogen for 1 year in a cryoprotective solution containing a low concentration of dimethyl sulfoxide and a high concentration of human serum (HuS). After thawing, the isolation, colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, cell senescence, apoptosis, and multi-lineage differentiation potential of hBM-MSCs were tested in media containing HuS compared with hBM-MSCs isolated from fresh fragments. Human BM-MSCs isolated from cryopreserved fragments expressed MSC markers until later passages, had a good proliferation rate, and exhibited the capacity to differentiate toward osteogenic, adipogenic, and myogenic lineages similar to hBM-MSCs isolated from fresh fragments. Moreover, the cryopreservation method did not induce cell senescence or cell death. These results imply that minimal processing may be adequate for the banking of tissue samples with no requirement for the immediate isolation and use of hBM-MSCs, thus limiting cost and the risk of contamination, and facilitating banking for clinical use. Furthermore, the use of HuS for cryopreservation and expansion/differentiation has the potential for clinical application in compliance with good manufacturing practice standards.

Stem cells isolated from human dental pulp and amniotic fluid improve skeletal muscle histopathology in mdx/SCID mice.

INTRODUCTION: Duchenne muscular dystrophy (DMD), caused by a lack of the functional structural protein dystrophin, leads to severe muscle degeneration where the patients are typically wheelchair-bound and die in their mid-twenties from cardiac or respiratory failure or both. The aim of this study was to investigate the potential of human dental pulp stem cells (hDPSCs) and human amniotic fluid stem cells (hAFSCs) to differentiate toward a skeletal myogenic lineage using several different protocols in order to determine the optimal conditions for achieving myogenic commitment and to subsequently evaluate their contribution in the improvement of the pathological features associated with dystrophic skeletal muscle when intramuscularly injected into mdx/SCID mice, an immune-compromised animal model of DMD. METHODS: Human DPSCs and AFSCs were differentiated toward myogenic lineage in vitro through the direct co-culture with a myogenic cell line (C2C12 cells) and through a preliminary demethylation treatment with 5-Aza-2'-deoxycytidine (5-Aza), respectively. The commitment and differentiation of both hDPSCs and hAFSCs were evaluated by immunofluorescence and Western blot analysis. Subsequently, hDPSCs and hAFSCs, preliminarily demethylated and pre-differentiated toward a myogenic lineage for 2 weeks, were injected into the dystrophic gastrocnemius muscles of mdx/SCID mice. After 1, 2, and 4 weeks, the gastrocnemius muscles were taken for immunofluorescence and histological analyses. RESULTS: Both populations of cells engrafted within the host muscle of mdx/SCID mice and through a paracrine effect promoted angiogenesis and reduced fibrosis, which eventually led to an improvement of the histopathology of the dystrophic muscle. CONCLUSION: This study shows that hAFSCs and hDPSCs represent potential sources of stem cells for translational strategies to improve the histopathology and potentially alleviate the muscle weakness in patients with DMD.

Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo.

Human dental pulp is a promising alternative source of stem cells for cell-based tissue engineering in regenerative medicine, for the easily recruitment with low invasivity for the patient and for the self-renewal and differentiation potential of cells. So far, in vitro culture of mesenchymal stem cells is usually based on supplementing culture and differentiation media with foetal calf serum (FCS). FCS is known to contain a great quantity of growth factors, and thus to promote cell attachment on plastic surface as well as expansion and differentiation. Nevertheless, FCS as an animal origin supplement may represent a potential means for disease transmission besides leading to a xenogenic immune response. Therefore, a significant interest is focused on investigating alternative supplements, in order to obtain a sufficient cell number for clinical application, avoiding the inconvenients of FCS use. In our study we have demonstrated that human serum (HS) is a suitable alternative to FCS, indeed its addition to culture medium induces a high hDPSCs proliferation rate and improves the in vitro osteogenic differentiation. Furthermore, hDPSCs-collagen constructs, pre-differentiated with HS-medium in vitro for 10 days, when implanted in immunocompromised rats, are able to restore critical size parietal bone defects. Therefore these data indicate that HS is a valid substitute for FCS to culture and differentiate in vitro hDPSCs in order to obtain a successful bone regeneration in vivo.

Fibroin scaffold repairs critical-size bone defects in vivo supported by human amniotic fluid and dental pulp stem cells.

The main aim of this study was the comparative evaluation of fibroin scaffolds combined with human stem cells, such as dental pulp stem cells (hDPSCs) and amniotic fluid stem cells (hAFSCs), used to repair critical-size cranial bone defects in immunocompromised rats. Two symmetric full-thickness cranial defects on each parietal region of rats have been replenished with silk fibroin scaffolds with or without preseeded stem cells addressed toward osteogenic lineage in vitro. Animals were euthanized after 4 weeks postoperatively and cranial tissue samples were taken for histological analysis. The presence of human cells in the new-formed bone was confirmed by confocal analysis with an antibody directed to a human mitochondrial protein. Fibroin scaffolds induced mature bone formation and defect correction, with higher bone amount produced by hAFSC-seeded scaffolds. Our findings demonstrated the strong potential of stem cells/fibroin bioengineered constructs for correcting large cranial defects in animal model and is likely a promising approach for the reconstruction of human large skeletal defects in craniofacial surgery.

Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix.

This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.