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。     

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.     

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.     

人体牙髓干细胞的分离与鉴定

目的探讨牙髓干细胞（DPSC）对牙周病,外伤及肿瘤等造成下颌骨缺损、口腔软组织与神经损伤的修复治疗作用。方法本研究利用组织块培养法分离出人体DPSC,用流式细胞仪进行了鉴定,并进行DPSC成骨、成脂、成神经的分化研究。结果分离出3株DPSC,流式细胞分析表明DPSC表达CD73和CD90标志物,但不表达生血干细胞标志物CD34。用茜素红染色表明DPSC能分化成骨细胞,油红O染色表明DPSC能分化成脂肪细胞,免疫免疫荧光染色表明DPSC分化的细胞表达神经细胞特异标志物TUJ1。结论组织块培养能够高效快速分离表达CD73和CD90的DPSC,在体外诱导条件下DPSC能分化为成骨细胞、脂肪细胞和神经细胞,此研究为DPSC在治疗和修复骨组织缺损和神经损伤中的临床应用提供了实验依据。     

Wnt signaling reprograms metabolism in dental pulp stem cells

Human dental pulp stem cells (DPSCs) can differentiate to a wide range of different cell lineages, and share some gene expression and functional similarities with pluripotent stem cells. The stemness of DPSCs can also be pharmacologically enhanced by the activation of canonical Wnt signaling. Here, we examined the metabolic profile of DPSCs during reprogramming linked to Wnt activation, by a short (48 hr) exposure to either the GSK3-β inhibitor BIO (6-bromoindirubin-3´-oxine) or human recombinant protein WNT-3A. Both treatments largely increased glucose consumption, and induced a gene overexpression of pyruvate and mitochondrial acetyl-coA producing enzymes, thus activating mitochondrial tricarboxylic acid cycle (TCA) metabolism in DPSCs. This ultimately led to an accumulation of reducing power and a mitochondrial hyperpolarization in DPSCs. Interestingly, Nile Red staining showed that lipid fuel reserves were being stored in Wnt-activated DPSCs. We associate this metabolic reprogramming with an energy-priming state allowing DPSCs to better respond to subsequent high demands of energy and biosynthesis metabolites for cellular growth. These results show that enhancement of the stemness of DPSCs by Wnt activation comes along with a profound metabolic remodeling, which is distinctly characterized by a crucial participation of mitochondrial metabolism.     

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.     

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".     

Effects of doxorubicin on human dental pulp cells in vitro

There is substantial information concerning the effects of continuous exposure to supratherapeutic or therapeutic concentrations of doxorubicin on human molar pulpal cells; the effects of continuous exposure to subtherapeutic concentrations of this agent are undetermined. To this end, we studied the proliferation of human fibroblasts and pulpal cells and their pattern of mineralized nodule deposition in vitro. Cell proliferation was assessed at 1, 3, 5, and 7 days from populations with either no exposure (control) or exposure to 10⁻⁶–10⁻⁹ mol/L doxorubicin. Mineralized nodule deposition and calcium-45 incorporation were assessed at 7 and 21 days of culture. Data were compared by factorial ANOVA and a post-hoc Tukey test. 10⁻⁶ and 10⁻⁷ mol/L doxorubicin significantly reduced the total number of viable pulpal cells in cultures from days 1 to 3 (p < 0.05); doxorubicin 10⁻⁶–10⁻⁹ mol/L significantly inhibited cell proliferation (p < 0.05) and DNA synthesis 5 days after plating (p < 0.001). After 21 days, doxorubicin 10⁻⁶–10⁻⁸ mol/L significantly decreased calcium-45 incorporation into pulpal cultures (p < 0.001); all dilutions significantly reduced the number of mineralized nodules within the 21-day pulpal cultures (p < 0.05). In addition, all dilutions of doxorubicin significantly inhibited fibroblast cell proliferation and incorporation of [³H]thymidine. In contrast, the fibroblast cultures did not produce mineralized nodules, suggesting that the mineralized nodules within the pulpal cell cultures did not result from dystrophic calcification. Thus, exposure to subtheraputic doxorubicin concentrations has potential adverse effects on mineralized tissue formation within the pulp, which could affect the rates of reparative dentin deposition within the tooth pulps of patients receiving this chemotherapeutic agent.     

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.     

Cellular response to orthodontically-induced short-term hypoxia in dental pulp cells

Orthodontic force application is well known to induce sterile inflammation, which is initially caused by the compression of blood vessels in tooth-supporting apparatus. The reaction of periodontal ligament cells to mechanical loading has been thoroughly investigated, whereas knowledge on tissue reactions of the dental pulp is rather limited. The aim of the present trial is to analyze the effect of orthodontic treatment on the induction and cellular regulation of intra-pulpal hypoxia. To investigate the effect of orthodontic force on dental pulp cells, which results in circulatory disturbances within the dental pulp, we used a rat model for the immunohistochemical analysis of the accumulation of hypoxia-inducible factor-1α in the initial phase of orthodontic tooth movement. To further examine the regulatory role of circulatory disturbances and hypoxic conditions, we analyze isolated dental pulp cells from human teeth with regard to their specific reaction under hypoxic conditions by means of flow cytometry, immunoblot, ELISA and real-time PCR on markers (Hif-1α, VEGF, Cox-2, IL-6, IL-8, ROS, p65). In vivo experiments showed the induction of hypoxia in dental pulp after orthodontic tooth movement. The induction of oxidative stress in human dental pulp cells showed up-regulation of the pro-inflammatory and angiogenic genes Cox-2, VEGF, IL-6 and IL-8. The present data suggest that orthodontic tooth movement affects dental pulp circulation by hypoxia, which leads to an inflammatory response inside treated teeth. Therefore, pulp tissue may be expected to undergo a remodeling process after tooth movement.     

Stage-specific embryonic antigen-4 identifies human dental pulp stem cells

Embryonic stem cell-associated antigens are expressed in a variety of adult stem cells as well as embryonic stem cells. In the present study, we investigated whether stage-specific embryonic antigen (SSEA)-4 can be used to isolate dental pulp (DP) stem cells. DP cells showed plastic adherence, specific surface antigen expression, and multipotent differentiation potential, similar to mesenchymal stem cells (MSC). SSEA-4+ cells were found in cultured DP cells in vitro as well as in DP tissue in vivo. Flow cytometric analysis demonstrated that 45.5% of the DP cells were SSEA-4+. When the DP cells were cultured in the presence of all-trans-retinoic acid, marked downregulation of SSEA-3 and SSEA-4 and the upregulation of SSEA-1 were observed. SSEA-4+ DP cells showed a greater telomere length and a higher growth rate compared to ungated and SSEA-4- cells. A clonal assay demonstrated that 65.5% of the SSEA-4+ DP cells had osteogenic potential, and the SSEA-4+ clonal DP cells showed multilineage differentiation potential toward osteoblasts, chondrocytes, and neurons in vitro. In addition, the SSEA-4+ DP cells had the capacity to form ectopic bone in vivo. Thus, our results suggest that SSEA-4 is a specific cell surface antigen that can be used to identify DP stem cells.     

Nell-1 attenuates lipopolysaccharide-induced inflammation in human dental pulp cells

Journal of Molecular Histology - Nel-like molecule type 1 (Nell-1) is a secreted protein that plays an important role in osteoinduction in multiple animal models. A previous study has suggested the...     

Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.     

Clastogenic activity of seven endodontic medications used in dental practice in human dental pulp cells

Numerous and varied chemical agents are used for endodontic treatments in dental practice. Endodontic medications are administered directly to the teeth in relatively high concentrations and chemical agents applied to enamel or dentin can penetrate the dental pulp tissue and circulate through the body in the bloodstream. In the present study, to assess safety regarding mutagenicity, we investigated the ability of seven endodontic medications to induce chromosome aberrations in human dental pulp cells. Chromosome aberrations were induced in cells treated with each of six endodontic medications, eugenol, guaiacol, modified phenol, phenol, thymol, and zinc oxide. The other endodontic medication, zinc chloride, failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by any of the endodontic medications tested. Our results indicate that the endodontic medications that exhibited a positive response are potentially mutagenic to human cells.