FGF8辅助牙源性上皮诱导hDPSCs分化为成牙本质细胞及牙髓细胞

目的:研究成纤维细胞生长因子8(FGF8)对成人牙髓干细胞(hDPSCs)定向分化为成牙本质细胞及牙髓组织的影响。方法:首先分离、克隆培养hDPSCs,通过流式细胞术检测细胞表面标志物鉴定hDPSCs;矿化液中添加50μg/L的FGF8诱导hDPSCs分化,通过real-time PCR检测分化后的细胞中牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、骨涎蛋白(BSP)和核心结合因子α1(Cbfa-1)在mRNA水平的表达;E11.5小鼠牙源性上皮联合FGF8与hDPSCs细胞团重组,再将组织块种植于裸鼠肾囊膜下培养,通过DNA原位杂交鉴定成牙本质细胞及牙髓细胞的来源。结果:成功分离培养hDPSCs,其表面标志物CD29和CD90呈阳性表达;经FGF8诱导的hDPSCs形成较明显的矿化结节,并且牙本质特异性蛋白DSPP、BSP及Cbfa-1表达量上调;E11.5小鼠牙源性上皮联合FGF8可以诱导hDPSCs分化为成牙本质细胞及牙髓细胞。结论:FGF8能够辅助牙源性上皮定向诱导hDPSCs分化为成牙本质细胞及牙髓细胞,并形成牙本质及牙髓腔结构。     

TGF-beta latency-associated peptides (LAPs) in human dentin matrix and pulp

Transforming growth factor (TGF)-beta s in dentin matrix provide a pool of bioactive molecules, but association with latency-associated peptides (LAPs) may influence their activity. We investigated TGF-beta 1, -beta 2, and -beta 3 LAP expression in sound and carious human teeth. Teeth were fixed and processed immediately following extraction prior to staining with rabbit polyclonal antibodies to the TGF-beta LAPs. A soluble dentin matrix fraction was prepared from dissected human dentin and sequential extraction of pulpal ECM was performed prior to purification. Fractions were Western blotted and probed with the LAP antibodies. All three LAPs were present in odontoblasts, cells of the pulp, and predentin; however, no staining of mineralized dentin matrix was seen. Similar patterns of expression were seen in carious tissue. Expression of TGF-beta LAPs in cells and pulpal matrix of healthy and carious teeth will be important in regulation of TGF-beta activity and may modulate the tissue response to injury.     

TGF-β Latency-Associated Peptides (LAPs) in Human Dentin Matrix and Pulp

Transforming growth factor (TGF)- &#103 s in dentin matrix provide a pool of bioactive molecules, but association with latency-associated peptides (LAPs) may influence their activity. We investigated TGF- &#103 1, - &#103 2, and - &#103 3 LAP expression in sound and carious human teeth. Teeth were fixed and processed immediately following extraction prior to staining with rabbit polyclonal antibodies to the TGF- &#103 LAPs. A soluble dentin matrix fraction was prepared from dissected human dentin and sequential extraction of pulpal ECM was performed prior to purification. Fractions were Western blotted and probed with the LAP antibodies. All three LAPs were present in odontoblasts, cells of the pulp, and predentin; however, no staining of mineralized dentin matrix was seen. Similar patterns of expression were seen in carious tissue. Expression of TGF- &#103 LAPs in cells and pulpal matrix of healthy and carious teeth will be important in regulation of TGF- &#103 activity and may modulate the tissue response to injury.     

Alteration in the expression of heat shock protein (Hsp) 25-immunoreactivity in the dental pulp of rat molars following tooth replantation.

The regeneration process of dental pulp following tooth replantation in rat molars was investigated by immunocytochemistry for heat shock protein (Hsp) 25 and protein gene product 9.5 (PGP 9.5). In control teeth at postnatal 4 weeks, the odontoblasts showed intense Hsp 25-immunoreactivity in the coronal dental pulp, but little or no immunoreactivity in the root and floor pulp. In contrast, the Hsp 25-negative odontoblasts in the latter areas displayed immunoreactivity for PGP 9.5. Tooth replantation caused loss of Hsp 25- and PGP 9.5-immunoreactions in the dental pulp during postoperative days 1-3. At postoperative day 5, plump cells with clear nucleoli and several fine processes--presumably newly differentiated odontoblasts--at the pulp-dentin border became immunopositive for Hsp 25. These data suggest that the expression of Hsp 25- and PGP 9.5-immunoreactivity reflects the status of differentiation of the odontoblasts. Furthermore, some pulpal nerve fibers as well as the Schwann cells in the dental pulp, ordinarily negative in Hsp 25-immunoreaction, acquired their immunoreactivity by postoperative day 5, but lost it thereafter, suggesting the involvement of Hsp 25 in the regeneration of pulpal nerve fibers. In the case of bone-like tissue formation in the pulp space, on the other hand, no Hsp 25-immunoreactive odontoblasts were recognized in the pulp-dentin border. Thus, the alignment of Hsp 25-immunopositive odontoblasts along the pulp-dentin border indicates a decisive factor for inducing the reparative dentin formation after tooth replantation.     

Cloned 3T6 Cell Line from CD-1 Mouse Fetal Molar Dental Papillae

Only primary pulpal cell cultures and one virally transformed mouse cell culture have been formally reported in the literature to synthesize proteins such as phosphophoryn which are unique to dentin matrix. In the present study, a mixed culture was derived from dental papilla cells of 18-19 fetal day CD-1 mouse mandibular first molars, maintained on a 3T6 plating regimen, and subsequently cloned after 28 passages. This cloned cell line (MDPC-23) exhibited several unique features, some of which were characteristic of odontoblasts in viva The features of this cell line included (1) epithelioid morphology of all cells with multiple cell membrane processes, (2) high alkaline phosphatase activity in all cells, (3) formation of multilayered nodules and multilayered cultures when maintained in ascorbic acid and β-glycerophosphate, and (4) expression of two markers for odontoblast differentiation, i.e. dentin phosphoprotein and dentin sialoprotein.     

Possible mechanisms of induction of dentinogenesis

The following hypothesis is proposed: The inner enamel epithelium influences all cells of the dental papilla very early during embryologic development--long before any morphologic changes due to histo-differentiation are noticeable. During normal odontogenesis, the epithelium acts as a second order inducer for the differentiation of odontoblasts and their subsequent production of dentin. Other mechanisms exist which can induce responding fibroblasts of the dental pulp to produce dentin or dentin-like tissue. Collagen matrix can act as such an inducer.     

Nestin expression in embryonic and adult human teeth under normal and pathological conditions

Nestin is an intermediate filament most related to neurofilaments and expressed predominantly in the developing nervous system and muscles. In the present study we examined the in vivo distribution of nestin in human teeth during embryonic development and in permanent teeth under normal and pathological conditions. The results show that nestin is first expressed at the bell stage and that its distribution is restricted in pulpal cells located at the cusp area of the fetal teeth. In young permanent teeth, nestin is found only in functional odontoblasts, which produce the hard tissue matrix of dentin. Expression is progressively down-regulated and nestin is absent from older permanent teeth. In carious and injured teeth, nestin expression is up-regulated in a selective manner in odontoblasts surrounding the injury site, showing a link between tissue repair competence and nestin up-regulation under pathological conditions. In an in vitro assay system of human dental pulp explants, nestin is up-regulated after local application of bone morphogenic protein-4. A similar effect is seen in cultures of primary pulp cells during their differentiation into odontoblasts. Taken together, these results suggest that nestin plays a potential role in odontoblast differentiation during normal and pathological conditions and that bone morphogenic protein-4 is involved in nestin up-regulation.     

Odontoblasts in odontogenic tumors

Odontoblasts are secretory cells displaying epithelial and mesenchymal features, which exist in a monolayer at the interface between the dentin and pulp of a tooth. During embryogenesis, these cells form a dentin shell and throughout life continue to produce dentin while, also acting as sensor cells helping to mediate tooth sensitivity. In this process, odontoblasts are forced to migrate inwards, resulting in an ongoing loss of pulp volume. Correspondingly, there is also a decrease in the surface area of the dentin which supports the odontoblast cell layer. As these events transpire, odontoblasts maintain a tightly controlled monolayer relationship to each other as well as to their dentin substrate. Stability is maintained laterally by epithelial attachment structures and transversely by complex cytoplasmic extensions into the supporting dentin. As a result, it is not possible for the layer to buckle to relieve the mechanical stresses, which develop during the inward migration. A theoretical consequence of this distinctive self-generated niche is the development of long term compressive stresses within the odontoblast population. We present a mechanobiology model, which causally relates the increase in cellular compressive stresses to contact inhibition of proliferation. We link this hypothesis to the observation that there are no reports of pulpal odontoblasts showing neoplasia or acquisition of changes suggestive of a pre-neoplastic phenotype.     

Immunocytochemical localization of the neurokinin 1 receptor in rat dental pulp

The dentin-pulp complex is a peripheral end-organ supplied by dense sensory nerve fibers. Substance P, a representative neuropeptide widely distributed in the dental pulp, has been reported to play roles in pain transmission and the amplification of inflammation. We analyzed here the expression of the neurokinin 1 (NK1) receptor, preferentially activated by substance P, using immunocytochemistry in rat dental pulp at both the light and electron microscopic levels. Conspicuous NK1 receptor immunoreactivity was found in the odontoblasts; immunolabelings were present at their plasma membrane and endosomal structures, especially in their cytoplasmic processes. Immunoreactions for NK1 receptor were also detectable in a part of the nerve terminals associated with the cytoplasmic processes of the odontoblasts. Furthermore, the endothelial cells of capillaries and post-capillary venules and the fibroblasts were labeled with the NK1 receptor in the subodontoblast layer. These findings suggest that pulpal cells and nerve fibers are targets for substance P that mediate multiple functions, including a vasoactive function and the regulation of vascular permeability as well as the modulation of pain transmission.     

Immunohistochemical localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp

Summary The distribution in oral tissues of endothelin, a multifunctional peptide originally identified within endothelial cells, and subsequently in some epithelial cells, neurons and neuroendocrine cells, has not been investigated yet. We have studied the localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp by immunohistochemical techniques. Such immunoreactivity was detected only within endothelial cells in both mature dental pulp and developing tooth. Arteries and veins of various sizes as well as small thin vessels displayed endothelin-like immunoreactivity. In the tooth germ, the cells of the enamel organ or the precursors of the odontoblasts were found unreactive. In the mature pulp, no cells of the stroma or nerves displayed endothelin-like immunoreactivity. These findings suggest that vascular endothelium may be the only source of endothelin in human dental tissues. It is tentatively proposed that endothelin released in mature tooth pulp may participate in the regulation of the pulpal blood flow. Although the possible role of endothelin in developing tissues is far from being clear, the mitogenic effects and the proto-oncogenes expression induced by endothelin in some cells raise the possibility that this peptide might also play a role during tooth development.     

Expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice

Differentiated odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. Preodontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated odontoblasts. In conclusion, nestin is valuable as a differentiation marker for odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.     

Mineralization and expression of Col1a1-3.6GFP transgene in primary dental pulp culture

We have examined and compared the effects of various differentiation-inducing media on mineralization, cell morphology and expression of pOBCol3.6GFP (3.6-GFP) in primary dental pulp cultures derived from 3.6-GFP transgenic mice. Our results show that media containing ascorbic acid only could not induce mineralization in primary dental pulp cultures. On the other hand, media containing ascorbic acid and beta-glycerophosphate induced formation of mineralized matrix-containing dentin. The amount of mineralized matrix was increased by addition of dexamethasone. Cells treated with ascorbic acid and beta-glycerophosphate were fibroblast like and cells treated with dexamethasone were cuboidal. In all culture conditions, high levels of 3.6-GFP were expressed in areas of mineralization.     

Tributyltin alters osteocalcin, matrix metalloproteinase 20 and dentin sialophosphoprotein gene expression in mineralizing mouse embryonic tooth in vitro

We showed in a previous in vitro study that tributyltin (TBT) arrests dentin mineralization and enamel formation in developing mouse tooth. The present aim was to investigate the effect of TBT on the expression of genes associated with mineralization of dental hard tissues. Embryonic day 18 mouse mandibular first molars were cultured for 3, 5 or 7 days and exposed to 1.0 μM TBT and studied by real-time quantitative polymerase chain reaction (RT-QPCR) for the expressions of osteocalcin (Ocn), alkaline phosphatase (Alpl), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and matrix metalloproteinase 20 (Mmp-20).Ocn, Mmp-20 and Dspp, whose expressions showed changes in RT- QPCR, were further analyzed by in situ hybridization of tissue sections. In situ hybridization showed that TBT decreased Ocn expression in odontoblasts but increased the expression in the epithelial tooth compartment. In QPCR assays, the net effect in the whole tooth was increased expression. TBT also reduced Mmp-20 expression in ameloblasts and odontoblasts. Dspp expression varied but both QPCR assays and in situ hybridization showed a decreasing trend. TBT exposure had no clear effect on Alpl and Dmp1 expressions. Increased Ocn expression by epithelial enamel organ may inhibit dentin mineralization and enamel formation. Decreased Ocn, Mmp-20 and Dspp expressions in odontoblasts may indicate delayed cell differentiation, or TBT may specifically decrease the expression of genes involved in dentin mineralization. While decreased Mmp-20 expression by TBT in ameloblasts may impair enamel mineralization, the coincident reduction in Mmp-20 and Dspp expressions in odontoblasts may potentiate the delay of dentin mineralization.     

Demonstration of amelogenin in the enamel-free cusps of rat molar tooth germs: immunofluorescent and immunoelectron microscopic studies.

The enamel-free cusps of 1-4 day-old rat mandibular first molars were investigated using the monoclonal antibody En3 against rat amelogenin at light and electron microscopic levels in order to clarify whether the enamel-free cusp is virtually devoid of enamel. At 1 day after birth, there were presecretory ameloblast-like cells (PALCs), which were short and were not polarized, at the cusp tips. They were close to the outer enamel epithelium. Hematoxylin positive enamel matrix was not distinctly observed in the enamel-free cusp by light microscopy, but almost continuous immunofluorescence for amelogenin was detected at the interface between PALCs and dentin. The penetration of immunopositive material toward the dental pulp was also observed in the enamel-free cusp. At 4 day after birth, both in the frontal section and in the horizontal section, almost continuous immunofluorescence was recognized at the interface between PALCs and dentin in the enamel-free cusp. The penetration of amelogenin toward the dental pulp was not seen in the enamel-free cusp. By immunoelectron microscopy, immunolabelling was recognized in the Golgi apparatus of PALCs, in a layer of amorphous material at the interface between PALCs and dentin, and in stippled material-like substance in the intercellular space between PALCs. Although no basement membrane was observed beneath PALCs, they did not have Tomes' processes. These investigations suggest that PALCs in the enamel-free cusp differentiate into the secretory cells and that they can synthesize and secrete the amorphous material containing amelogenin at the interface between PALCs and dentin. The penetration of amelogenin toward the dental pulp might play a role in the interaction between PALCs and odontoblasts in the enamel-free cusp and/or the initiation of mineralization of predentin.     

The effect of glucocorticosteroid treatment on dentine formation in the Lewis rat, a histological study

Glucocorticosteroids are widely used in the treatment of chronic illnesses and have been reported to cause premature obliteration of the pulp space. During the active stages of dentinogenesis, odontoblasts are growth hormone receptor (GHr) positive. The aims of this study were to determine if the glucocorticosteroid, prednisone, affected the rate of dentine deposition and odontoblast expression of GHr in the rat molar. Following subcutaneous injection of 0.05 mg/kg, 1.0 mg/kg or 5.0 mg/kg prednisone for 20 days, immature and mature molars from rats aged 3 and 6 weeks respectively, were examined histologically. Distribution of GHr expression was determined immunohistochemically. No morphological differences were observed in molars from prednisone treated animals. Prednisone did not appear to enhance dentine deposition in immature molars but in mature molars significantly increased dentine deposition on the roof of the pulp chamber at a dosage of 5.0 mg/kg (p < 0.001). In all immature molars, odontoblasts and pulp cells expressed GHr immunoreactivity. In mature molars, odontoblasts and pulpal cells from controls did not show GHr immunoreactivity. However, odontoblasts and pulp cells were GHr immunoreactive in mature molars from animals treated with prednisone.     

Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro

The adult dental pulp is innervated by sensory trigeminal axons and efferent sympathetic axons. Rat trigeminal ganglia extend neurites when co-cultivated in vitro with pulpal tissue explants, suggesting that pulpal cells secrete soluble molecules that stimulate the growth of trigeminal ganglion axons. In addition, cultured pulpal cells produce mRNAs for neurotrophins and glial cell line-derived neurotrophic factor-family members. These data suggest that neurotrophic factors are involved in the formation of a pulpal innervation. Here, we examine how pulpal cells and 3T3 fibroblasts overexpressing certain neurotrophic factors (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, glial cell line-derived neurotrophic factor or neurturin) influence survival and growth of single trigeminal ganglion neurones in vitro in quantitative terms. The results show that most of the neurotrophic factor-overexpressing fibroblasts induce similar neuronal soma diameters, but higher survival rates and neurite lengths compared with pulpal cells. With respect to neurite growth pattern, trigeminal ganglion neurones co-cultured with fibroblasts overexpressing nerve growth factor develop a geometry that is most similar to that seen in co-cultures with pulpal cells. We conclude that none of the fibroblasts overexpressing neurotrophic factors can fully mimic the effects of pulpal cells on trigeminal ganglion neurones, and that nerve growth factor promotes a neurite growth pattern most similar to the picture seen in co-cultures with pulpal cells.     

Effects of alginate hydrogels and TGF-beta 1 on human dental pulp repair in vitro

The objective of this study was to investigate the use of alginate hydrogels to present either exogenous or endogenous transforming growth factor (TGF)-beta 1 to the dentin-pulp complex to signal reparative processes. Hydrogels were prepared, applied to cultured human tooth slices and the effects on tertiary dentinogenesis examined histologically. Both TGF-beta 1-containing and acid-treated alginate hydrogels, but not untreated hydrogels, upregulated dentin matrix secretion and induced odontoblast-like cell differentiation with subsequent secretion of regular tubular dentin matrix on cut pulpal surfaces. It is concluded that TGF-beta 1 can signal both induction of odontoblast-like cell differentiation and upregulation of their matrix secretion in the human dentin-pulp complex. Alginate hydrogels provide an appropriate matrix in which dental regeneration can take place and may also be useful for delivery of growth factors, including TGF-beta s, to enhance the natural regenerative capacity of the dental pulp.