Dentinogenic potential of human adult dental pulp cells during the extended primary culture

Despite the frequent use of primary dental pulp cells in dental regenerative research, few systematic studies of stemness for osteogenic and dentinogenic differentiation of human adult pulp cells have been reported. To investigate the stemness of human adult dental pulp cells, pulp tissues were obtained from extracted third molars and used as a source of pulp cells. In FACS analysis and immunophenotyping, the general mesenchymal stem cell markers CD44, CD90, and CD146 were highly expressed in early passages of the pulp cell culture. The stem cell population was dramatically decreased in an expansion culture of human dental pulp cells. When pulp cells were treated with additives such as β-glycerophosphate, ascorbic acid, and dexamethasone, nodule formation was facilitated and mineralization occurred within 2 weeks. Expression of osteogenic markers such as alkaline phosphatase, osteocalcin, and osteonectin was relatively low in undifferentiated cells, but increased significantly under differentiation conditions in whole passages. Dentinogenic markers such as dentin sialophosphoprotein and dentin matrix protein-1 appeared to decrease in their expression with increasing passage number; however, peak levels of expression occurred at around passage 5. These data suggested that stem cells with differentiation potential might exist in the dental pulp primary culture, and that their phenotypes were changed during expansion culture over 8-9 passages. Under these conditions, a dentinogenic population of pulp cells occurred in limited early passages, whereas osteogenic cells occurred throughout the whole passage range.     

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.     

Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells

Background information. Although adult bone‐marrow‐derived cell populations have been used to make teeth when recombined with embryonic oral epithelium, the differences between dental and non‐dental stem‐cell‐mediated odontogenesis remain an open question. Results. STRO‐1⁺ (stromal precursor cell marker) DPSCs (dental pulp stem cells) and BMSSCs (bone marrow stromal stem cells) were isolated from rat dental pulp and bone marrow respectively by magnetic‐activated cell‐sorting techniques. Their odontogenic capacity was compared under the same inductive microenvironment produced by ABCs (apical bud cells) from 2‐day‐old rat incisors. Co‐cultured DPSCs/ABCs in vitro showed more active odontogenic differentiation ability than mixed BMSSCs/ABCs, as indicated by the accelerated matrix mineralization, up‐regulated alkaline phosphatase activity, cell‐cycle modification, and the expression of tooth‐specific proteins and genes. After cultured for 14 days in the renal capsules of rat hosts, recombined DPSC/ABC pellets formed typical tooth‐shaped tissues with balanced amelogenesis and dentinogenesis, whereas BMSSC/ABC recombinants developed into atypical dentin—pulp complexes without enamel formation. DPSC and BMSSC pellets in vivo produced osteodentin‐like structures and fibrous connective tissues respectively. Conclusions. DPSCs presented more striking odontogenic capability than BMSSCs under the induction of postnatal ABCs. This report provides critical insights into the selection of candidate cells for tooth regeneration between dental and non‐dental stem cell populations.     

Increased resorptive activity and accompanying morphological alterations in osteoclasts derived from the oim/oim mouse model of osteogenesis imperfecta

The current study addresses whether alterations in osteoclasts (OCs) derived from oim/oim mice, an established model of moderate-to-severe OI, are present. Bone marrow cells from oim/oim and wildtype (+/+) mice were cultured on bone slices in the presence of MCSF and RANKL and evaluated at days 0, 1, 2, 4, and 7. OCs were identified by tartrate-resistant acid phosphatase (TRAP) staining, and bone slice resorption pits were analyzed by reflection microscopy. Flow cytometry was used to examine CD51 (integrin alphaV) and CD61 (integrin beta3) markers. Confocal microscopy was used to assess changes in OC morphology and resorption. There was no difference between the OC precursors of the two genotypes in expression of CD51 and CD61 markers. At day 2, the bone slices seeded with oim/oim cells had a greater percentage of mononuclear cells associated with resorption pits compared to +/+ bone slices. At day 4, the diameter and area of oim/oim OCs were larger compared to the +/+ OCs, and the number of nuclei per OC was also greater for the oim/oim group. At day 7, the oim/oim OCs contained more F-actin rings compared to the +/+ OCs, and the number of OCs in the oim/oim group was greater compared to the +/+ group. The resorbed area of bone slices for the oim/oim group was also greater compared to the +/+ group at day 7. In conclusion, oim/oim mononuclear resorbing cells and OCs showed cellular changes and greater resorptive activity compared to +/+ cells, features that likely contribute to dysregulated bone remodeling in OI.     

A simple method for the quantitation of the stem cells derived from human exfoliated deciduous teeth using a luminescent cell viability assay

Stem cells from human exfoliated deciduous teeth are a population of highly proliferative postnatal stem cells and have been characterized as multipotent stem cells. In this study we developed a fast and sensitive method for stem cells derived from human exfoliated deciduous teeth count, using a luminescent viability assay. We isolated stem cells from normal exfoliated deciduous teeth using collagenase/dispase digestions solutions. Separated stem cells were placed in opaque-walled multiwall plates in culture alpha Modified Eagle’s Medium. For dental pulp stem cells quantitation we used a simple method for determining the number of viable cells based on ATP concentration. Cells attached to the bottom of the multiwall plates were counted with the luminescent assay and were cultured for mesenchymal markers expression. Moreover cells attached to the bottom of the multiwall plates were directed toward the osteogenic, adipogenic, lineages at the respective passages. Flow cytometry was used for immunophenotyping of cultured dental stem cells from exfoliated deciduous teeth. Cells that were counted with the luminescent assay, after culture formed fibroblastic morphology and were expressed the mesenchymal stem cell markers CD29, CD105, CD146, CD44. There was a correlation between the number of cells plated for culture and the number of mesenchymal stem cells after culture. Osteogenic and adipogenic differentiation of the cells counted with the luminescent assay was performed. The luminescent signal of viable mesenchymal dental stem cells isolated from dental pulp of exfoliated teeth represents an ideal method for mesenchymal stem cells count before culturing.     

A substractive PCR-based cDNA library from human odontoblast cells: identification of novel genes expressed in tooth forming cells.

Odontoblasts are highly specialized cells aligned at the edge of the dental pulp. As a step towards understanding the complex mechanisms underlying their terminal differentiation, the gene expression pattern was examined in human cultured odontoblast cells. Suppression substractive hybridization (SSH) was used to establish a substracted cDNA library specific for human odontoblasts. For this purpose, cDNAs from human cultured fibroblastic pulp cells were substracted to cDNA from human cultured odontoblasts. The nucleotide sequence of 154 substracted cDNA clones was determined. We identified 130 preferentially expressed gene fragments in odontoblasts as compared with the fibroblastic pulp cells. Ten of them were already identified in odontoblasts such as DSPP, BSP, enamelysin and Col1A1. We confirmed their overexpression by RT-PCR on the cultured cells and in vivo by in situ hybridization on human molars. Another 64 clones corresponded to known genes. Among them, two clones were of particular interest: reelin, which was first detected in the brain and osteoadherin, which was first located in bone. Fifty-six clones were unknown genes even though 82% matched expressed sequence tags or genomic clones. A reverse Northern dot blot showed that 96% of them were overexpressed at different rates in cultured odontoblasts. These latest results indicate that there are still unknown genes that are associated with the control of the odontoblast phenotype. Thus, cloning of odontoblast differentiation-associated genes not only opens up new methods of elucidating the normal development but also the recruitment of odontoblasts when required to initiate repair of dentin.     

Human platelet lysate enhances the proliferative activity of cultured human fibroblast-like cells from different tissues

Several studies have shown the presence of fibroblast-like cells in the stromal fraction of different tissues with a high proliferative and differentiation potential. Platelet alpha granules contain growth factors released into the environment during activation. The effects of different supplements for culture medium (human serum, bovine serum and platelet lysate) on cultured human fibroblast-like cells from bone marrow, adipose tissue, trabecular bone and dental pulp have been compared. Expression of typical stromal and hematopoietic markers was analyzed and proliferative rates were determined. Flow cytofluorometry showed a homogenous pattern in serial-passaged cells, with a high level of stromal cell-associated markers (CD13, CD90, CD105). The presence of platelet lysate in culture media increased the number of cell generations obtained regardless of cell source. This effect was serum-dependent. Cell-based therapies can benefit by the use of products from human origin for “ex vivo” expansion of multipotent cells.     

Osteoclast differentiation gene expression profiling reveals chemokine CCL4 mediates RANKL‐induced osteoclast migration and invasion via PI3K pathway

The migration of osteoclasts (OCs) from circulation and bone marrow into bone surface plays a critical role in the pathogenesis of some bone resorptive diseases, such as rheumatoid arthritis and osteoporosis. To date, how the migration of OCs remains unclear. We investigated gene expression profiling in osteoclastic differentiation of bone marrow–derived macrophages (BMMs) into OCs by microarray analysis. We identified 387 genes overexpressed in osteoclastic differentiation of BMMs. Among them, chemokine CCL4 showed a robust up‐regulation signal. High expression of CCL4 was validated in primary BMMs and OC precursor cell line RAW264.7 during differentiation into OCs. The CCL4 neutralization decreased RANKL‐induced OC precursor cell migration and invasion in Matrigel‐coated transwell membranes assay and in vitro wound healing assay. However, CCL4 inhibition did not affect OCs differentiation and differentiation associated gene expression. The CCL4 inhibition promoted the PI3K phosphorylation at 45 to 60 minutes after RANKL stimulation in RAW264.7. This study indicated that chemokine CCL4 is an important regulator for OCs migration via PI3K pathway, providing a novel therapy target for bone resorptive diseases.     

基于PI3K/AKT通路探究上调miR-210对牙髓干细胞增殖、凋亡能力的影响

摘要 目的：研究基于磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)通路探究上调微小RNA 210(miR-210)对大鼠牙髓干细胞增殖、凋亡能力的影响。方法：选取10只健康Sprague-Dawley（SD）雄性大鼠,颈椎脱臼处死后提取大鼠下切牙牙髓,进行牙髓干细胞培养和鉴定。分为正常组（未进行处理）,miR-210抑制组（给予20 nmol/L的miR-210抑制物）,miR-210对照组（给予20 nmol/L的miR-210模拟物）三组。采用CCK-8法检测牙髓干细胞增殖活性,酶联免疫吸附试验（ELISA）检测ALP活性,流式细胞仪检测细胞凋亡,采用免疫印迹（Western blot）检测PI3K、AKT蛋白。结果：与正常组相比,miR-210抑制组细胞增殖、ALP活性降低,细胞凋亡率升高;miR-210对照组细胞增殖、ALP活性升高,细胞凋亡率降低（P＜0.05）。与miR-210抑制组相比,miR-210对照组细胞增殖、ALP活性升高,细胞凋亡率降低（P＜0.05）。与正常组相比,miR-210抑制组PI3K、p-AKT蛋白表达降低,miR-210对照组PI3K、p-AKT蛋白表达升高（P＜0.05）。与miR-210抑制组相比,miR-210对照组PI3K、p-AKT蛋白表达升高（P＜0.05）。结论：miR-210通过调控PI3K、p-AKT蛋白激活PI3K/AKT通路,促进大鼠牙髓干细胞增殖,抑制牙髓干细胞凋亡。     

An approachable human adult stem cell source for hard-tissue engineering

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/CD34+/STRO-1+ cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10(-8) mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies.     

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。     

Neural crest stem cell property of apical pulp cells derived from human developing tooth

Recent reports have described that NCSCs (neural crest-derived stem cells) are not only present in the embryonic neural crest but also in the adult tissues. Dental pulp is one of mesenchymal soft tissues origin from cranial neural crest cells, and thought to be a source of adult stem cells. Here, we investigated the existence of NCSC-like cells in apical pulp of human developing tooth. Human impacted third molars with immature apex freshly extracted were obtained. The cells derived from the apical pulp tissue not framed by dentin or the coronal pulp tissues were cultured by primary explant culture. APDCs (apical pulp-derived cells) and CPCs (coronal pulp cells) formed spheres under neurosphere culture condition. The number of spheres from APDCs was larger than that from CPCs. The sphere-forming cells derived from APDCs had self-renewal capacity, and expressed neural crest-associated markers (p75, Snail and Slug) and NSC (neural stem cell) markers (Nestin and Musashi1). The expression pattern of mesenchymal stem cell markers, CD105 and CD166, on the surface of sphere-forming cells derived APDCs was different from that of APDCs. These sphere-forming cells could differentiate into multiple mesenchymal lineages (osteoblasts, adipocytes, chondrocytes and smooth muscle cells) and neural lineage (neurons) in vitro, and generated ectopic bone tissues on the border of HA (hydroxyapatite) scaffold in vivo. The results of this study suggest that APDCs contain cells with characteristics of NCSCs reported previously in mice. Humans developing tooth with immature apex is an effective source of cells for neural crest lineage tissue regeneration.     

Histochemical and immunocytochemical study of hard tissue formation in dental pulp during the healing process in rat molars after tooth replantation

Dental pulp is assumed to possess the capacity to elaborate both bone and dentin matrix under the pathological conditions following tooth injury. This study was undertaken to clarify the mechanism inducing bone formation in the dental pulp by investigating the pulpal healing process, after tooth replantation, by micro-computed tomography (μ-CT), immunocytochemistry for heat-shock protein (HSP)-25 and cathepsin K (CK), and histochemistry for both alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Under deep anesthesia, the upper right first molar of 4-week-old Wistar rats was extracted and immediately repositioned in the original socket. In control teeth at this age, the periphery of the coronal dental pulp showed intense ALP-positive and HSP-25-positive reactions, whereas there were no TRAP-positive or CK-positive cells. Tooth replantation weakened or terminated ALP-positive and HSP-25-positive reactions in the pulp tissue at the initial stages. At 3–7 days after operation, the ALP-positive region recovered from the root apex to the coronal pulp followed by HSP-25-positive reactions in successful cases showing tertiary dentin formation. In other cases, TRAP-positive and CK-positive cells appeared in the pulp tissue of the replanted tooth at postoperative days 5–10 and remained associated with the bone tissue after 12–60 days. Immunoelectron microscopy clearly demonstrated that CK-positive osteoclast-lineage cells made contact with mesenchymal cells with prominent nucleoli and well-developed cell organelles. These data suggest that the appearance of TRAP-positive and CK-positive cells is involved in the induction of bone tissue formation in dental pulp.This work was supported in part by a grant from MEXT to promote 2001-multidisciplinary research project (in 2001–2005) and by KAKENHI (B) from MEXT, Japan (no. 16390523 to H.O.).     

Different expressions of connexin 43 and 32 in the fibroblasts of human dental pulp

The expression and localization of gap junctional proteins connexin (Cx) 26, 32, and 43 was examined in human dental pulp. Dental pulp tissues were obtained from human third molars immediately after extraction. Some pulp tissues were used for cell culture, and the rest for histological observations. Immunostaining for cultured dental pulp fibroblasts (DPFs) showed that Cx32 and 43 were expressed in human DPFs, and proteins corresponding to 27 (Cx32) and 43kDa (Cx43) were identified by Western blot analysis. Immunostaining for tissue sections showed that the expression of Cx32 and 43 was observed in the entire region of the pulp and further strong expression of Cx32 was established beneath the cell-rich zone. Considering the close relationship between Cx types and cell functions, the results indicate that DPFs beneath the cell-rich zone may have specific, Cx32-related functions. The cell rich zone is thought to contain progenitor odontoblasts that can be induced to differentiate into mature odontoblasts in response to wounding. Therefore, it may be hypothesized that DPFs just beneath the cell-rich zone produce proteins and induce odontoblast differentiation from the cells in the cell-rich zone.     

TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)-beta superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-beta1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-beta3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-beta3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-beta3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-beta3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-beta3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-beta3 soaked beads in vivo. These results indicate for the first time that TGF-beta3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.