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PubMed2005

A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (LAB).

UNLABELLED: Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. A new c-kit+/CD34+/CD45- cell population of stromal bone producing cells (SBP/DPSCs) was selected, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and then in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue, which is markedly positive for several bone antibodies. This tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone-containing osteocytes. INTRODUCTION: Recently it has been reported that human dental pulp stem cells (DPSCs) are detectable, in humans, only up to the age of 30 years and that they are able to produce in vitro only sporadic calcified nodules and to form, after transplantation in vivo, a mineralized tissue. MATERIALS AND METHODS: Stem cells, derived from human adult dental pulp of healthy subjects 30-45 years of age, were cultured, and cells were selected using a FACSorter. Light microscope, histochemistry, immunofluorescence, and RT-PCR analyses were performed to study both stem and differentiating cells. RESULTS AND CONCLUSIONS: A new c-kit+/CD34+/CD45- cell population of stromal bone producing cells (SBP/DPSCs) has been selected by FACSorting, expanded, and cultured. These SBP/DPSCs are highly clonogenic and, in culture, differentiate into osteoblast precursors (CD44+/RUNX-2+), still capable of self-renewing, and in osteoblasts, producing, in vitro, a living autologous fibrous bone (LAB) tissue. This new-formed tissue is markedly positive for several antibodies for bone, including osteonectin, bone sialoprotein, osteocalcin, fibronectin, collagen III, and bone alkaline phosphatase (BALP). Cells producing LAB can be stored at -80 degrees C for a long period of time and are an extraordinary source of osteoblasts and mineralized fibrous bone tissue. In this study, we also showed that, in aged humans, stem cells can be detected from their pulps. The produced LAB is a fibrous bone tissue resembling the human bone during mineralization, with an external layer formed by osteoblasts markedly positive for osteocalcin. This newly formed tissue constitute an ideal source of osteoblasts and mineralized tissue for bone regeneration. In fact, after in vivo transplantation into immunocompromised rats, LAB formed lamellar bone containing osteocytes.

Adult↗
PubMed2002

Interleukin-1beta activity and collagen synthesis in human dental pulp fibroblasts.

Immunopathologic reactions play a significant role in inflammatory diseases of dental pulp. Interleukin-1beta (IL-1beta) is recognized as a key player in mediating cellular immune response. In this study, we measured the content of IL-1beta and its effect on collagen synthesis in cultures of fibroblasts derived from healthy and diseased dental pulps. We found that diseased pulp fibroblasts contain 2.5-fold greater amounts of IL-1beta and synthesized 80% greater amounts of collagen compared with healthy pulp fibroblasts. However, exogenous IL-1beta failed to stimulate collagen synthesis by diseased fibroblasts, whereas collagen synthesis by healthy pulp fibroblasts was stimulated by more than 2-fold. These observations imply that pulp disease induces abnormalities associated with fibroblast response toward IL-1beta.

Analysis of Variance↗
PubMed1989

An experimental study of osteogenesis in explants of rat dental pulp, periodontal ligament, bone marrow and muscle cells in vitro.

Cells of dental pulp, periodontal ligament, bone marrow and muscle derived from the same rat were cultured in vitro. Outgrowth of cells from these four explants were examined using phase-contrast microscope, light microscope and scanning electron microscope. Furthermore, cell lengths were measured randomly from SEM photographs and growth curves were plotted by calculating cell numbers. These cells had different morphology in both shape and length. The bone marrow cells showed the most rapid proliferation rate followed by muscle, periodontal ligament and most slowly in dental pulp cells.

Animals↗
PubMed2000

Failure of presynaptic purinoceptors to modulate noradrenaline release from sympathetic nerves in human dental pulp.

The effects of putative presynaptic P1- and/or P2-purinoceptors on the release of noradrenaline from sympathetic nerves in human dental pulp were examined by testing the effects of agonists and an antagonist of these receptors on the stimulation-induced overflow of [(3)H]noradrenaline from tissue treated with desipramine (0.3 micromol/l) and preincubated with [(3)H]noradrenaline (0.6 micromol/l). The P1-purinoceptor agonists adenosine (1.0 mmol/l) and 2-chloroadenosine (0.01-1.0 mmol/l) and the antagonist 8-cyclopentyl-1,3-dipropyl xanthine (1.0 micromol/l), and the P2-purinoceptor agonists ATP (0.1 mmol/l) and beta, gamma-methylene-ATP (0.01 mmol/l), did not modulate the release of noradrenaline. Adenosine was also without effect in dental pulp treated with the alpha(2)-adrenoceptor antagonist rauwolscine. It is concluded that presynaptic P1-purinoceptors and those P2-purinoceptors activated by adenine nucleotides are either not present on sympathetic nerves in human dental pulp or that they exert little or no effect on the release of noradrenaline.

2-Chloroadenosine↗
PubMed2006

In vitro bone production using stem cells derived from human dental pulp.

To harvest bone for autologous grafting is a daily problem encountered by craniofacial and oral surgeons. Stem cells derived from human dental pulp are able to differentiate in osteoblasts and are a potential source of autologous bone produced in vitro. The authors describe their preliminary results in this new field with its potential application in craniomaxillofacial surgery. Dental pulp was gently extracted from 34 human permanent teeth (all third molars) of patients 19 to 37 years of age. After they were digested, the cells were selected using a cytometer for c-kit, STRO-1, CD34, CD45, and then for CD44 and RUNX-2. This study, made on a considerable number of cases, provided evidence that dental pulp is extremely rich in stem cells, which were c-kit+/CD34+/STRO-1+/CD45-, capable of differentiation toward several stromal-derived differentiated cells and mainly osteoblasts. These findings, supported by the large number of cases, are of great interest for tissue regeneration, tissue-based clinical therapies, and transplantation.

Adolescent↗
PubMed1999

Macrophages, dendritic cells and T lymphocytes in rat buccal mucosa and dental pulp following 5-fluorouracil treatment.

Cancer chemotherapeutic drugs may affect immunocompetent cells of oral soft tissues, causing an impaired capacity to induce immune defence reactions. This study was designed to investigate changes in the number of macrophages, dendritic cells and T lymphocytes in the oral mucosa and dental pulp following treatment with the antineoplastic agent 5-fluorouracil (5-FU). Rats were given 5-FU (30 mg/kg or 50 mg/kg) i.v. on days 0, 1, 2, 5, 6 and 7. The number of cells in buccal epithelium and dental pulp expressing ED2, MHC class II, or CD2 molecules was analyzed following immunohistochemical peroxidase staining. Major histocompatibility complex (MHC) class II molecules were analyzed in epithelial sheets and in epithelial cell suspensions by flow cytometry. Increasing concentrations of 5-FU changed the morphology of the epithelial Langerhans cells with a reduced dendritic appearance as the most prominent feature. At 50 mg/kg of 5-FU, the oral epithelium detached from the connective tissue at the basement membrane. MHC class II molecule-expressing cells were reduced in number in the lamina propria of the buccal mucosa and in the dental pulp after both low and high dose of 5-FU, but only after high dose in the epithelium. The number of ED2- and CD2-expressing cells in the dental pulp was only slightly reduced by 5-FU treatment at both low and high dose, while these cells decreased in number in the oral mucosa. The varying sensitivity to 5-FU by macrophages, dendritic cells, and T cells depending on the tissues in which they reside may be due to differences in cell origin or differences in antigenic load.

Animals↗
PubMed2002

Developmental changes and regional differences in histochemical localization of hyaluronan and versican in postnatal molar dental pulp.

AIM: The main aim of this study was to investigate the developmental changes in the distribution patterns of hyaluronan (HA) and versican in postnatal rat molar dental pulp, in order to confirm the hypothesis that the distribution of both molecules can vary with physiological conditions in the dental pulp. METHODOLOGY: Thirty postnatal Sprague-Dawley rats, 1, 7, 14, 21, 28, 35, 42 and 49 days old, were used for this study. Immunohistochemistry for versican with monoclonal antibodies 12C5 and CS-56 and histochemical staining for HA with HA-binding protein were applied to paraffin sections of the mandibular first molars at each age. RESULTS: At day 1, both molecules were evenly distributed in the interior parts of the pulp, but strong reactions for both molecules appeared in the subodontoblastic layer of the coronal pulp by the completion of crown formation. However, a strong reaction for HA and a weak reaction for versican were seen in the subodontoblastic layer of the radicular pulp. Furthermore, a versican-deficient, low-HA area first appeared in the interior of the coronal pulp at day 42 and expanded at day 49. CONCLUSIONS: Distribution of hyaluronan and versican in the dental pulp varied with age and also showed regional differences between the coronal and the radicular pulp, and this supports the hypothesis described above.

Age Factors↗
PubMed2003

Catalase activity in human healthy and inflamed dental pulps.

AIM: To examine the catalase activity in clinically healthy and symptomatic human dental pulps to verify if an active defence system against oxidizing agents is present as a response to bacterial invasion. METHODOLOGY: Thirty-three systemically healthy patients, 18 females and 15 males (ages: 11.0-25.9 years; mean 18.8 +/- 3.6), were the source of the pulp tissue. The condition of the pulps was assessed using clinical and radiographic evaluations. The specimens were recovered by longitudinally grooving and splitting the teeth (if extracted) or during endodontic treatment, and were matched for age and sex between the healthy and inflamed specimen groups. Catalase activity was determined through spectrophotometric methods. RESULTS: Enzymatic activity was 1126 +/- 343 and 3074 +/- 698 mU mL(-1) x mg of total protein in the healthy and inflamed pulp tissue specimens, respectively. This difference was statistically significant (P<0.01). CONCLUSIONS: These results indicate a role for catalase during dental pulp inflammation in humans, and may represent an inherent biological defence system against reactive oxidants of this tissue.

Adolescent↗
PubMed1990

A clonal prostaglandin-responsive cell line (RDP 4-1) derived from rat dental pulp.

The RDP 4-1 cell line was established from isolated rat dental pulp and cloned with limiting dilution in 96-multiwell plates. Despite their fibroblast-like appearance, RDP 4-1 cells required more than 5% (v/v) heat-inactivated fetal calf serum to maintain the cell growth and function. Type I collagen, but not type II and type III collagen, was immunocytochemically detected in the cells in subconfluent cultures. Prostaglandin E2 (PGE2, 5 ng/ml-5 micrograms/ml) dose-dependently increased cAMP accumulation, while prostaglandin F2 alpha (PGF2 alpha, 5 micrograms/ml) slightly increased it, in the presence of 0.1 mM isobutylmethyl xanthine. PGE2 (5 ng/ml-5 micrograms/ml) also stimulated phosphoinositide hydrolysis to produce inositol phosphates (IPs) in a time- and dose-dependent manner in the presence of 10 mM LiCl. PGF2 alpha was a more potent stimulator of phosphoinositide hydrolysis. Both prostaglandins increased cytoplasmic free calcium, independent of the level of extracellular calcium. Prolonged exposure (48 h) of the cells to PGE2 at higher concentrations (greater than or equal to 0.5 micrograms/ml) increased DNA synthesis. In these RDP 4-1 cells, thus, PGE2 receptors appeared to be associated with both cAMP and IPs-Ca2+ signaling systems, and that PGF2 alpha receptors are poor stimulators of the former but more potent ones of the latter. Our results suggest that both prostaglandins have some roles in regulating dental pulp metabolism.

Animals↗
PubMed2003

[The roles of Smad 2/3 in transforming growth factor-beta 1 signaling in human dental pulp cells].

OBJECTIVE: To explore the roles of Smad 2/3 in transforming growth factor-beta(1) (TGF-beta(1)) signaling by human dental pulp cells. METHODS: Laser scanning confocal microscope was used to observe translocation of Smad 2/3 from plasma into nucleus in cultured dental pulp cells at early stage of TGF-beta(1) treatment, and changes of Smad 2/3 protein expression at later stage were evaluated by Western blot analyses. RESULTS: The expression of Smad 2/3 (fluorescence intensity) kept decreasing in cytoplasm but increasing in nucleus within 2 h after TGF-beta(1) treatment, forming a trend that Smad 2/3 translocated into nucleus from cytoplasma. The total amount of Smad 2 protein remained unchanged before and after TGF-beta(1) treatment, but the expression level of Smad 3 decreased markedly after 24 h treatment and kept dropping by 48 h. CONCLUSIONS: The results suggest that the Smad 2/3 may be the downstream signal transducers of TGF-beta(1) in human dental pulp cells and Smad 2/3 may mediate TGF-beta(1) signaling by translocation early in TGF-beta(1) treatment, while down-regulation of Smad 3 expression by TGF-beta(1) at later stage is involved in negative modulation of TGF-beta(1) signaling.

Adolescent↗
PubMed2006

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation.

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.

Dental Pulp↗
PubMed1984

Neuropeptide Y: occurrence and distribution in dental pulps.

Nerve fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in the dental pulp of several mammals, including man. Generally, the NPY fibers were more numerous in the apical part than in the coronal part and were distributed around small blood vessels and as single fibers in the pulpal stroma. Sequential staining with antibodies against the enzyme dopamine-beta-hydroxylase (DBH), a marker for adrenergic neurons, and NPY showed that DBH and NPY were located in the same perivascular nerve fibers. Further, since chemical and surgical sympathectomy caused the disappearance of pulpal NPY fibers, it is conceivable that NPY fibers in the dental pulp are identical with adrenergic ones. There is thus a morphological basis for suggesting that NPY and noradrenaline cooperate in regulating pulpal blood flow.

Adrenergic Fibers↗
PubMed1999

Tooth movement and vascularity of the dental pulp: a pilot study.

The effect of orthodontic tooth movement on the dental pulp was assessed histologically in twelve subjects. The participants in this study required the extraction of at least two maxillary first premolars for orthodontic treatment. They were asked to wear a maxillary removable appliance that acted to move a randomly determined premolar in a buccal direction. The appliance was designed to avoid contacting the contra-lateral tooth that was used as the matched control. The appliance was initially worn for a week to ensure patient comfort and cooperation. The appliance was then activated and the patient dismissed. After two weeks, the appliance was reactivated. Both the control and experimental teeth were extracted three weeks later, on the thirty-fifth day of activated appliance wear. The teeth were fixed, decalcified and sectioned. The sections were stained with haematoxylin and eosin for histological examination. This investigation demonstrated that orthodontic tooth movement did have an effect upon the dental pulp, causing vasodilation in the pulp of an orthodontically stressed tooth.

Activator Appliances↗
PubMed1996

Osteotropic factor-stimulated synthesis of thrombospondin in rat dental pulp cells.

The amount of thrombospondin (TSP) mRNA in confluent clonal rat dental pulp cells was increased by transforming growth factor-beta (TGF-beta), 1 alpha-25-dihydroxyvitamin D3 (1,25(OH)2D3), and phorbol 12,13-didecanoate (PDD), with maximal levels 6, 36 and 2 h, respectively, after stimulation. These increases were accompanied by enhanced syntheses of TSP proteins which were found in the different forms in cell layer/matrix fraction (198 and 165 kDa TSP) and the culture medium (180 kDa TSP). These three factors also raised the mRNA level of osteopontin, which is thought to play an important role in mineralization in dentin and bone. The order of effectiveness of these factors was PDD > TGF-beta > 1,25(OH)2D3 for all the stimulations described above. These results suggest that the osteotropic factors enhance TSP synthesis at the pretranslational level and that TSP produced by dental pulp cells participates in formation of reparative dentin.

Animals↗
PubMed2000

Prostaglandin E2 enhances bradykinin-evoked iCGRP release in bovine dental pulp.

Mediators produced during inflammation are responsible for hyperalgesia and expression of neurotransmitters and receptors in the nervous system. The production of bradykinin (BK) and the prostaglandins (PGs) may regulate initiation of pain. This study tested the hypothesis that BK and prostaglandin E2 (PGE2) have a positive interaction in evoking neurosecretion of immunoreactive calcitonin gene-related peptide (iCGRP). Bovine dental pulp was prepared and stimulated by the superfusion method with BK alone and in combination with PGE2. Kinin receptor antagonists to bradykinin-evoked release of iCGRP were also tested. Also tested was the hypothesis that dental pulp contains either the B1 or B2 or both BK receptors. Results showed that PGE2 enhanced BK-evoked iCGRP release by more than 50%. Western immunoblots revealed detectable B2 receptor protein with no detectable B1 receptor protein. We conclude that BK evokes iCGRP release from bovine dental pulp which is enhanced by a positive interaction with PGE2. Neurosecretion is evoked from isolated terminals of dental pulp fibers via the bradykinin B2 receptor-dependent mechanism.

Analysis of Variance↗
PubMed1998

Responses of macrophage-associated antigen-expressing cells in the dental pulp of rat molars to experimental tooth replantation.

Bacterial infection of the dental pulp is a major hindrance to successful pulp regeneration after tooth replantation. This study examined how macrophages and class II molecule-expressing cells of the pulp respond to tooth replantation, on the hypothesis that they contribute to the defence and repair of the traumatized pulp. Upper right first molars of 5-week-old male Wistar rats were replanted immediately after extraction; contralateral untreated teeth served as controls. Pulpal cells expressing macrophage-associated antigens were immunohistochemically demonstrated at 0 h (immediately after the replantation) to 84 days postoperatively using antirat monoclonal antibodies OX6 (anti-class II molecules), ED1 (pan-macrophage antibody, reactive also with dendritic cells) and ED2 (anti-resident macrophages). Between 3 and 7 days postoperatively, ED1+ and OX6+ cells, but not ED2+ cells, were concentrated in areas of degeneration formed in the coronal pulp, and frequently showed a marked accumulation along the pulp-dentine border of the cuspal area. Confocal laser scanning microscopy revealed that some of the OX6+ cells with a dendritic profile extended several cytoplasmic processes into the dentinal tubules communicating with the enamel-free area at the tip of the cusp. From 14-84 days, approx. two-thirds of specimens exhibited pulp-tissue regeneration with increasing formation of reparative dentine. Following the formation of sound reparative dentine, cells positive to each antibody were distributed more centrally in the pulp than in the controls, and thus did not show any accumulation along the pulp-dentine border. However, in the other specimens where a bone-like hard tissue had formed in the pulp chamber, many ED1+ and OX6+ cells were still concentrated in the remaining pulp tissue and showed a marked accumulation along the pulp dentine border. Few ED2+ cells were observed in these specimens. These findings suggest that, following tooth replantation, exudative macrophages are actively engaged in eliminating dentinal tubule-derived infectious stimuli and that class II molecule-expressing cells, most probably containing dendritic cells, are positioned strategically at the outermost portion of the injured pulp to monitor incoming antigens. The intensity of the pulpal defence reaction may be dependent on the status of hard-tissue formation, which influences the amount of incoming antigens.

Animals↗
PubMedDate not supplied

Local application of IGF1 on dental pulp mechanically exposed; in vivo study on rabbit.

IGF1 (Insulin Growth Factor, 1) was intentionally applied onto pulp tissues, aiming to provoque a dentine regeneration process through the stimulation of the dentinoblasts' potententials. 72 cavities were hence performed on rabbit molars, intentionally exposing the dental pulp. Different concentrations of IGF1 were then applied; The histo and anatomo-pathological observations showed persistent vitality of the pulp without any sign of necrosis, even 6 weeks after the IGF1 application. Dentinoblasts layers (as an indication of the regeneration activity) were counted, according to a pre-established protocol, at days 7, 14, 22, 28 and 42. The type of the applied IGF1, was carefully selected to be "Binding Protein Resistant" (IGF-BPR), so to avoid any inhibition of the IGF1 action by the endogenous binding proteins (Hochscheid and coll). The results were conclusive in indicating the IGF1 as an efficient dental pulp capping product.

Animals↗
PubMed1997

Osteocalcin expression in young and aged dental pulps as determined by RT-PCR.

Dental pulps were obtained from third molars of young adults (17-25 yr) or from molar teeth of individuals > 50 yr of age and examined for the expression of osteocalcin (OC) mRNA by RT-PCR. OC was selected as a determinant of pulp vitality, because it has long been associated with the production of hard tissue matrix in teeth and bone. For comparative purposes, the expression of OC in each pulp was normalized relative to its housekeeping gene-product GAPDH by the establishment of a OC/GAPDH ratio. This study demonstrated that OC expression, presumably by cells of odontoblast lineage, does not diminish relative to the extant cell population. Our findings suggest, despite a reduction in volume and cell numbers, that the pulps of aging teeth retain a capacity for dentin deposition and a potential for caries and trauma resistance.

Adolescent↗