Enzyme separation techniques for the study of growth of cells from layers of bovine dental pulp

Summary Effects of the enzymes trypsin, papain, bromelains and ficin in bovine dental pulp tissue were studied. Minced or whole pulps were subjected to each enzyme at 17°, 20° and 37°C for set time intervals, after which aliquots of supernatant fluid were removed for cell counts and viability tests. Pooled samples were subsequently cultured as monolayers in Eagle’s MEM plus 10% calf serum. The dissociation characteristics were quite distinct for each enzyme, although quite similar between minced and whole pulp. A parallel histological study was made of the residual pulp tissue. Ficin was found to be the most suitable enzyme for future studies on the growth of isolated pulp cells from various layers of the bovine pulp, due to its even rate of cell removal, and the good initial viability and subsequent growth of the separated cells in monolayer culture. Further studies on ficin may show that it is more suitable for enzymatic separation of tissues generally than the more commonly used trypsin, a major advantage being its use in media containing Ca²⁺ and Mg²⁺. Supported in part by NIH Grant No. DE 02908 and by United Health Foundation Summer Fellowships to J.T.F. and J.F.C. Supported in part by N.I.H. Grant 5 R01-DE 02908 to W.A.M. and by United Health Foundation Summer Fellowships to J.T.F. and J.F.C.     

Stem cells of dental pulp

Any clinician dreams to obtain the regeneration of the destroyed organ for his patient. In the human being, the regeneration of complex structures is not possible, except the liver and the bone marrow, which can be regenerated because of the presence of adult stem cells in these tissues. The stem cells have two principal properties: they ensure their self-renewal and they have the ability to differentiate into several cellular types. Using specific markers allowing the identification of the stem cells in bone marrow, stem cells were observed in dental pulp tissues. Although the origin, the identification, and the localization of these stem cells of dental pulp remain under consideration, the optimism in research on stem cells permits to believe that the knowledge on dental stem cells will lead to their use in therapeutics.     

Synthesis of procollagen by dental pulp cells from incisor in vitro.

Fibroblast-like cells were isolated from dental pulp of rabbit incisor, and were shown to synthesize and secrete procollagen. Analysis of the secreted procollagen and their CNBr peptides indicated that the prominent form was type I procollagen. Our results also suggested the presence of type III procollagen as a minor component synthesized by pulp cells in vitro.     

Purification and characterization of bovine dental pulp collagenase inhibitor

Root pulps from bovine unerupted wisdom teeth produce a potent collagenase inhibitor together with latent collagenase when cultured in Eagle's minimal essential medium (Biochem. Int. 5, 763, 1982). The inhibitor was purified more than 700-fold from the explant medium using Con A-Sepharose, Ultrogel AcA 44 and DE-52 cellulose columns. It showed a single band (MW = 36,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but showed multiple bands on basic (pH 8.3) polyacrylamide gel electrophoresis and electrofocusing. The inhibitor is a sialo-glycoprotein containing approx. 20% carbohydrate by weight and its composition suggests that it contains complex-type oligosaccharides. The electrophoretic heterogeneity of the inhibitor was proved to be due to the attachment of different numbers of sialic acid residues. All the SH groups were demonstrated to exist as six disulfide linkages which might be involved in the inhibitory activity. The bovine pulp inhibitor does not combine with collagen. The addition of the inhibitor to activated collagenase resulted in dose-dependent inhibition of the enzyme activity, but the interaction between the inhibitor and activated collagenase is not tight enough for the complex to remain intact during gel filtration column chromatography. A rabbit antiserum was prepared against the inhibitor, and immunoglobulin purified from the antiserum can completely abolish the inhibitory activity of the inhibitor.     

Scaling-up of dental pulp stem cells isolated from multiple niches

Dental pulp (DP) can be extracted from child's primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2-5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3-4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2'-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their "stemness".     

Isolation of pluripotent stem cells from human third molar dental pulp

Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.     

牙髓干细胞的研究进展

牙髓干细胞是来源于牙髓组织中的一种成体干细胞,该种细胞具有高度增殖、自我更新的能力和多向分化潜能。牙髓干细胞的研究对牙髓再生、牙体修复等牙组织工程将产生重要的意义。该文就牙髓干细胞的研究现状作一综述,并对其应用前景进行探讨。     

N-acetylcysteine protects dental pulp stromal cells from HEMA-induced apoptosis by inducing differentiation of the cells

Resin-based materials are now widely used in dental restorations. Although the use of these materials is aesthetically appealing to patients, it carries the risk of local and systemic adverse effects. The potential risks are direct damage to the cells and induction of immune-based hypersensitivity reactions. Dental pulp stromal cells (DPSCs) and oral keratinocytes are the major cell types which may come in contact with dental resins such as 2-hydroxyethyl methacrylate (HEMA) after dental restorations. Here we show that N-acetylcysteine (NAC) inhibits HEMA-induced apoptotic cell death and restores the function of DPSCs and oral epithelial cells. NAC inhibits HEMA-mediated toxicity through induction of differentiation in DPSCs, because the genes for dentin sialoprotein, osteopontin (OPN), osteocalcin, and alkaline phosphatase, which are induced during differentiation, are also induced by NAC. Unlike NAC, vitamins E and C, which are known antioxidant compounds, failed to prevent either HEMA-mediated cell death or the decrease in VEGF secretion by human DPSCs. More importantly, when added either alone or in combination with HEMA, vitamin E and vitamin C did not increase the gene expression for OPN, and in addition vitamin E inhibited the protective effect of NAC on DPSCs. NAC inhibited the HEMA-mediated decrease in NF-kappaB activity, thus providing a survival mechanism for the cells. Overall, the studies reported in this paper indicate that undifferentiated DPSCs have exquisite sensitivity to HEMA-induced cell death, and their differentiation in response to NAC resulted in an increased NF-kappaB activity, which might have provided the basis for their increased protection from HEMA-mediated functional loss and cell death.     

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

Background  

Chimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.     

Allogenic banking of dental pulp stem cells for innovative therapeutics

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.     

Determination of age in the Japanese monkey from growth layers in the dental cementum

The teeth of 14 Japanese monkeys (Macaca fuscata) were examined to establish an exact method of determining age by histological observation of dental cementum. The cementum showed annual growth layers, which were especially remarkable in the incisor root and in the molar cementum deposited at the junction of the roots. The layer of cementum formed in winter appears as a dark layer in stained sections and as a translucent layer in unstained ground sections. In the incisor the first dark and light layers are formed at the age of three years, whereas in the molar they do not appear at a definite age. The layers are thick and clear in the upper medial incisor. As a result, the age of a Japanese monkey can be determined by adding two to the number of dark layers and an outer light layer. It is interesting that the formation of the cementum of the first molar begins a few years after its eruption. The relation between this fact and the pressure of occlusion is discussed.     

Peptidergic nerves in human dental pulp. An immunocytochemical study

The peptidergic innervation of human dental pulp was studied with indirect immunofluorescence and immunoperoxidase techniques. Pulpal nerve fibres displaying immunoreactivity for cholecystokinin, calcitonin gene-related peptide, C-terminal flanking peptide of neuropeptide tyrosine, leucine-enkephalin, methionine-enkephalin, neuropeptide K, neuropeptide tyrosine, peptide with N-terminal histidine and C-terminal isoleucine, somatostatin-28, substance P and vasoactive intestinal polypeptide were observed. Immunoreactive axon varicosities were detectable within radicular and coronal nerve trunks and within the nerve plexus of Raschkow in the para-odontoblastic region. Many peptidergic nerve fibres were observed in association with blood vessels of various sizes. Substance P- and calcitonin-gene-related peptide-immunoreactive axons were visible in the odontoblastic layer. The occurrence of VIP- and PHI-immunoreactive fibres lends support to the hypothesis that human tooth may be supplied by parasympathetic nerves. The immunocytochemical results here shown provide a morphological basis to previous experimental studies concerning the possible roles of neuropeptides in nociception mechanisms, control of the blood flow and modulation of the inflammatory response in dental tissues.     

Recruitment of dental pulp cells by dentine and pulp extracellular matrix components

The present study aimed to determine whether dentine tissue and preparations of extracellular matrix (ECM) from pulp (pECM) and dentine (dECM), and breakdown products, influenced pulp cell migration. Chemotaxis transwell and agarose spot assays demonstrated that both dentine and pulp ECM molecules acted as chemoattractants for primary pulp cells. Chemoattractant activities of dECM and pECM were enhanced when subjected to acid and enzymatic breakdown, respectively. This enhanced activity following physiologically relevant breakdown may be pertinent to the disease environment. Pulp cell migration in response to dental ECMs was dependent on an active rho pathway. Recruited cells exhibited increased stem cell marker expression indicating that dental ECMs and their breakdown products selectively attract progenitor cells that contribute to repair processes. In conclusion, combined these results indicate that ECM molecules contribute to cell recruitment necessary for regeneration of the dentine-pulp complex after injury.     

Senescence and odontoblastic differentiation of dental pulp cells

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.     

Effect of mechanical tension on the human dental pulp cells

In this study, we have evaluated the effects of mechanical tension on the proliferation and extracellular matrix (ECM) production of human dental pulp stem cells (DPSCs) using a flexwell system that imposed cyclic mechanical tension at 0.03 Hz with 0, 5, and 8% strains. In the early stage (4 days), DPSCs at 5 and 8% strains had a similar proliferation, which was higher than the control. However, in the late stage (10 days), DPSCs at 8% strain had a higher proliferation than the control and 5% strains. This result clearly demonstrated that DPSC proliferation under tension varied with culture time. In addition, mechanical tension was shown to increase the amount of lactate dehydrogenase (LDH) released during culture. RT-PCR analysis was used to show that mechanical tension also increased collagen and osteopontin expression and decreased α-smooth muscle actin (α-SMA) expression. Furthermore, FACS analysis showed that CD105 expression did not change in all groups but CD 90 expression decreased at 8% strain. In conclusion, our results suggest that an appropriate level of mechanical tension can serve as a potent positive modulator of proliferation, differentiation and ECM production in DPSCs.