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Isolation, characterization and investigation of differentiation potential of human periodontal ligament cells and dental follicle progenitor cells and their response to BMP-7 in vitro

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Abstract

The aim of this study was to assess the factors, mechanisms and the differences between periodontal ligament (PDL) cells and denta l follicle (DF) progenitor cells towards the osteoblastic/cementoblastic differentiation and to investigate the effects of BMP-7 on developmental (DF) and mature tissue-derived (PDL) cells, respectively. Primary cell culture of PDL cells and DF progenitor cells was performed. Osteogenic differentiation was evaluated using von Kossa, Alizarin Red S and immuno-histo-chemistry staining of osteocalcin. Gene expression pattern was evaluated via real-time PCR. A series of CD surface marks were tested using flow cytometry and fluorescence-activated cell-sorting analysis was performed. Real-time RT-PCR demonstrated similar gene expression pattern of PDL cells and DF progenitor cells: the expression of OPN and OCN significantly was elevated when incubated with osteogenic components, Runx2 was unaffected, and Osteorix was hardly expressed whether in basic medium or induction medium. In addition, BMP-7 induced osteoblast/cementoblast differentiation of PDLSCs and DF progenitor cells in a dose- and time-dependent manner, as reflected by enhanced Runx2 and (OCN) mRNA transcript expression. BMP-7 triggers PDL cells and DF progenitor cells to differentiate towards an osteoblast/cementoblast phenotype.

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... As a matter of fact, the furcation defects were almost filled with regenerative periodontal tissue in these two groups, supporting the effectiveness of BMP-7 loaded to the CS/β-GP hydrogel in promoting periodontal regeneration, which is in agreement with previous findings [10,11]. The potential of BMP-7 in cementogenic and osteogenic differentiation of periodontal ligament stem cells has been well documented [30,31], but its appropriate dose remains a complex issue because some studies found that high dose BMPs caused local tissue inflammation, bone resorption, and adipogenesis [32][33][34]. In this study, we chose a BMP-7 dose of 100 ng/mL because our previous study found that BMP-7 at this dose could promote differentiation of dental pulp stem cells [35]. ...
... In this study, we chose a BMP-7 dose of 100 ng/mL because our previous study found that BMP-7 at this dose could promote differentiation of dental pulp stem cells [35]. Similar doses haven been applied in other studies and been proven to enhance osteogenesis and promote differentiation of dental follicle progenitor cells to osteoblasts or cementoblasts [31,36,37]. Our in vivo results argue for the feasibility of this dose of BMP-7 in conjunction with the hydrogel. ...
... A literature survey could reveal that third molar teeth were mostly used for PDLSC isolation. Briefly [31], impacted third molars were surgically extracted, and periodontal dental ligament was gently scraped from the middle root surface. Coronal and apical portions of the ligament were not used in order to avoid contamination by gingival and pulpal cells. ...
... After digestion, tissue was seeded into culture flasks with alpha-modification of Eagle's Medium supplemented with 10% fetal bovine serum, 2-mM Glutamine, 100-U/mL penicillin and 100-μg/mL: streptomycin solution at 37°C in 5% CO 2 in a humidified atmosphere. After single cells were attached on the plastic bottom of the flask, non-adherent cells were removed by changing the medium [31]. ...
... This process involves various types of intracellular and intercellular signal transmission and pathways, which constitute a complex signal transduction network. 26 DFCs have the extraordinary ability to differentiate into different and diverse cell types. The periodontal tissues, such as the cementum, periodontal ligament and alveolar bone, all originate from dental follicles. ...
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... Though all of them behaved promoted effect on osteogenesis, the mechanisms and effect levels were distinctive. Both BMP2 and BMP7 mediated DFC osteoblast differentiation in a time and dose-dependent manner while others were not [1,71]. BMP2 and BMP4 were critical to the early stage of osteogenic differentiation while BMP6 functioned both in the early and late stages of this process. ...
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Dental follicle cells (DFCs) are a group of mesenchymal progenitor cells surrounding the tooth germ, responsible for cementum, periodontal ligament, and alveolar bone formation in tooth development. Cascades of signaling pathways and transcriptional factors in DFCs are involved in directing tooth eruption and tooth root morphogenesis. Substantial researches have been made to decipher multiple aspects of DFCs, including multilineage differentiation, senescence, and immunomodulatory ability. DFCs were proved to be multipotent progenitors with decent amplification, immunosuppressed and acquisition ability. They are able to differentiate into osteoblasts/cementoblasts, adipocytes, neuron-like cells, and so forth. The excellent properties of DFCs facilitated clinical application, as exemplified by bone tissue engineering, tooth root regeneration, and periodontium regeneration. Except for the oral and maxillofacial regeneration, DFCs were also expected to be applied in other tissues such as spinal cord defects (SCD), cardiomyocyte destruction. This article reviewed roles of DFCs in tooth development, their properties, and clinical application potentials, thus providing a novel guidance for tissue engineering.
... [21][22][23] Recently, mesenchymal stem cells (MSCs) from dental follicles were isolated and differentiated into clonogenic, plastic-adherent, fibroblast-like cells. 24,25 The multipotency of dental follicle stem cells (DFSCs) makes these cells potentially useful in the reconstruction of bone under inflammatory conditions. [26][27][28][29] As a disease that is frequently associated with tooth loss, periodontal disease is defined as the destruction of alveolar bone and periodontal tissues by bacterial infection. ...
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The complex microenvironment of the periodontal wound creates many challenges associated with multitissue regeneration of periodontal lesions. Recent characterization of mesenchymal stem cell-like populations residing in periodontal ligament tissues has shown that these cells exhibit features of postnatal stem cells. Despite these advances, a lack of consistency in design of preclinical studies and a limited study of allogeneic transplantation applications has restricted our understanding of their clinical utility in the treatment of periodontal disease. The aim of this study was to assess the regenerative potential of allogeneic periodontal ligament stem cells (PDLSCs) in a rat periodontal fenestration defect mode and to identify an optimal end time-point suitable for quantitative assessment of tissue regeneration. Periodontal fenestration defects, created in Sprague Dawley rats, were treated with allogeneic PDLSCs seeded onto Gelfoam(®) (Absorbable gelatin sponge; Pharmacia Corporation, Kalamazoo, MI, USA) or with Gelfoam(®) alone, or remained untreated. Experimental rats were killed at 7, 14, 21 or 28 d after surgery and the tissues were processed for immunohistochemical and histomorphometric examination. Defects treated with PDLSCs showed significantly greater percentage bone fill and length of new bone bridge compared with the untreated group or the group treated withGelfoam(®) alone on days 14 and 21. Similarly, a statistically significant difference was achieved within specimens retrieved on day 21 for analysis of regeneration of cementum/periodontal ligament (PDL)-like structures. The present investigation shows that allogeneic PDLSCs have a marked ability to repair periodontal defects by forming bone, PDL and cementum-like tissue in vivo. The results suggest that treatment periods of 14 and 21 d are optimal end time-points for quantitative assessment of periodontal regeneration within the rodent fenestration-defect model utilized in the present study.
Article
The bone-specific transcription factor, Cbfa1, regulates expression of the osteocalcin (OCN) gene and is essential for bone formation. However, little is known about the mechanisms regulating Cbfa1 activity. This work examines the role of the MAPK pathway in regulating Cbfa1-dependent transcription. Stimulation of MAPK by transfecting a constitutively active form of MEK1, MEK(SP), into MC3T3-E1 preosteoblast cells increased endogenous OCN mRNA, while a dominant negative mutant, MEK(DN), was inhibitory. MEK(SP) also stimulated activity of a 147-base pair minimal OCN promoter, and this stimulation required an intact copy of OSE2, the DNA binding site for Cbfa1. Effects of MEK(SP) were specific to Cbfa1-positive osteoblast-like cells. A purified His-tagged Cbfa1 fusion protein was directly phosphorylated by activated recombinant MAPK in vitro. Furthermore, 32P metabolic labeling studies demonstrated that MEK(SP) clearly enhanced phosphorylation of Cbfa1 in intact cells, while MEK(DN) decreased phosphorylation. The specific MEK1/MEK2 inhibitor, PD98059, inhibited extracellular matrix-dependent up-regulation of the OCN promoter, indicating that the MAPK pathway and, presumably, Cbfa1 phosphorylation are also required for responsiveness of osteoblasts to extracellular matrix signals. This study is the first demonstration that Cbfa1 is controlled by MAPKs and suggests that this pathway has an important role in the control of osteoblast-specific gene expression.
Article
The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2A/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-β1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfa1 may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype. J. Cell. Biochem. 73:114–125, 1999. © 1999 Wiley-Liss, Inc.
Article
Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor-β (TGF-β) superfamily, including TGF-βs themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-β superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-β1. The single application of relatively high doses of TGF-β1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF-β1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis. Anat Rec 259:97-107, 2000. © 2000 Wiley-Liss, Inc.
Article
Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP-2 on the in vitro and in vivo biologic activity of well-characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP-2. hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP-2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8-wk healing period. The effects of rhBMP-2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP-2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. In the present study, rhBMP-2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down-regulated following treatment with rhBMP-2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP-2 on cementum and PDL tissue regeneration by hPDLSCs.
Article
The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days, The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11), The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH, Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.
Article
Types I, V, and VI collagen were immunohistochemically localized in frozen and paraffin sections of human permanent teeth, periodontal ligament, and alveolar bone, by means of polyclonal antibodies. Hyaluronidase was effective in exposing epitopes of the various collagen types. The expression of type I collagen in predentin was strong in frozen sections, whereas the dental pulp stained relatively weakly. Staining intensity in the dentin matrix decreased toward enamel and cementum. Reactivity in the periodontal ligament was moderate, and it was weaker in the alveolar bone and also in cementum, which stained more intensely in paraffin sections. Staining for type V collagen was strong in the pulp. Weak reactivity in predentin became uniformly evident in frozen sections only, and dentin was negative. The periodontal ligament stained with moderate intensity, and a weak staining reaction was seen in cementum and bone. Staining for type VI collagen in the pulp and periodontal ligament was strong, whereas predentin and dentin were negative. The alveolar bone stained moderately, and non-uniform reactivity was present in cementum. In non-mineralized dental tissues, the use of frozen material enabled good immunohistochemical localization of the distinct collagen types to be carried out. Their distribution patterns in dental tissues not only differed, but the relative staining intensities for each collagen type in the pulp and predentin were inversely related. However, differences may exist in the exposure of the epitopes of collagen(s) between soft and mineralized tissues.
Article
This enzyme's specific activity was high in the periodontal ligament with other tissues in both species, but much higher in the rabbit. The alkaline phosphatase in the rabbit ligament is a liver/kidney/bone form and not an intestinal one.
Article
To study the relation between expression of the putative myofibroblast marker alpha-smooth muscle actin and the remodelling of extracellular matrix, immunocytochemical, gel electrophoresis, and collagen gel contraction studies were performed on two human fibroblast subtypes. Double immunolabelling for total actins and alpha-smooth muscle (sm) actin as well as affinity labelling of filamentous and monomeric actins in gingival fibroblasts demonstrated that alpha-sm was colocalized in stress fibres and in regions with high levels of monomeric actin throughout the cytoplasm. alpha-sm comprised up to 14% of total cellular actin as assessed by 2D gel electrophoresis. Thirteen different gingival and seven different periodontal ligament fibroblast lines constitutively expressed on alpha-sm actin. These cells exhibited up to 60% inter-line variations of fluorescence due to alpha-sm actin and up to 70% and 45% inter-line variation in the rate of collagen gel contraction. Quantitative, single cell fluorimetry of alpha-sm actin immunoreactivity demonstrated a linear relation between gel contraction and alpha-sm actin (correlation coefficients of 0.71 for gingival and 0.61 for periodontal ligament cells), but there was no detectable relationship between total actin content and gel contraction. In contrast, flow cytometry demonstrated that 99% of the total gated cells from cell lines exhibiting rapid gel contraction showed alpha-sm actin staining above background fluorescence as compared to only 35% of cells with slow rates of gel contraction. Contracting collagen gels stained with FITC-phalloidin showed cells with well-developed stress fibres that were progressively more compact and elongated during the time of maximal gel contraction. To examine the dependence of gel contraction on assembly of monomeric actin into actin filaments, cells were electroporated in the presence of phalloidin or cytochalasin D. Collagen gels exhibited up to 100% inhibition of gel contraction that was dose-dependent. Gel contraction was inhibited 93% by electroinjection of cells with alpha-sm actin antibody prior to incubation, but the antibody did not inhibit actin assembly after attachment and spreading on substrates. These data indicate that gel contraction is dependent on alpha-sm actin expression and that alpha-sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix.
Article
Fibroblasts are the predominant cells of the periodontal ligament (PL) and have important roles in the development, function, and regeneration of the tooth support apparatus. Biological processes initiated during the formation of the PL contribute to the long-lasting homeostasic properties exhibited by PL fibroblast populations. The formation of the PL is likely controlled by epithelial-mesenchymal and epithelial hard tissue interactions, but the actual mechanisms that contribute to the development of cellular lineages in the PL are unknown. Fibroblasts in the normally functioning PL migrate through the tissue along collagen fibres to cementum and bone and in an apico-coronal direction during tooth eruption. ADULT TISSUE: Cell kinetic experiments have shown that PL fibroblasts comprise a renewal cell system in steady-state and the progenitors can generate multiple types of more differentiated, specialized cells. Progenitor cell populations of the PL are enriched in locations adjacent to blood vessels and in contiguous endosteal spaces. In normally functioning periodontal tissues, there is a relatively modest turnover of cells in which apoptotic cell death balances proliferation. Large increases of cell formation and cell differentiation occur after application of orthodontic forces or wounding. As PL cells comprise multiple cellular phenotypes, it has been postulated that after wounding, the separate phenotypes repopulating the site will ultimately dictate the tissue form and type. PL fibroblasts play an essential role in responses to mechanical force loading of the tooth by remodelling and repairing effete or damaged matrix components. In consideration of the important roles played by fibroblasts in PL homeostasis, they could be described as "the architect, builder, and caretaker" of the periodontal ligament.
Article
Background: Cementum continues to be the least-known mineralized tissue. Although recent advances in the field of molecular biology have contributed to an understanding of the involvement of molecular factors in cementum formation during development and regeneration, cementogenesis on a cell biological basis is still poorly understood. Virtually nothing is known about cementoblast origin, differentiation, and the cell dynamics during normal development, repair, and regeneration. This review describes the recent findings of cementogenesis on roots of human premolars and opposes them to those of teeth from other mammals, particularly the rodent molar. Methods: Using light and electron microscopy, light microscopic radioautography, and various measurements, a comprehensive insight into the development and repair of cementum during and after root formation and tooth eruption has been achieved for human premolars. Results: Cementum is a highly responsive mineralized tissue. This biological activity is necessary for root integrity and for bringing and maintaining the tooth in its proper position. With regard to cementum formation and periodontal fiber attachment, considerable species-particularities exist that are mainly based on differences in growth rates and tooth sizes. Since root development and initial cementogenesis last on the average 5-7 years in human premolars, cementum formation in these teeth is characterized by along-lasting phase of prefunctional development, with occurs independent of principal periodontal fiber attachment to the root and which may take 5 years or more. The first molar of the rat, however, is in functional occlusion 3 1/2 weeks after the onset of root formation. Since initial cementum formation and periodontal fiber attachment to the root occur almost at the same time in this tooth, the distinction between cells associated with one or the other process is very difficult to achieve, and cementogenesis cannot be described independent of periodontal fiber attachment to the root. Therefore, the determination of cementoblast origin in the rodent molar may be intricate. Conclusions: Taking into account these species differences, the current description on the origin and differentiation of cementoblasts is inconsistent and the description of cementogenesis is still incomplete. This review calls into question the currently held concept of cementogenesis and offers a possible alternative.
Article
Monoclonal antibodies (McAbs) against the surface of osteoblastic cells have been used to characterize the osteogenic lineage. In view of the paucity of probes against the surface of normal human osteogenic cells, we sought to generate McAbs which could be used for both in vivo and in vitro studies. We raised a series of McAbs against early osteoblastic cell surface antigens by immunizing mice with human mesenchymal stem cells (MSCs) that had been directed into the osteogenic lineage in vitro. After screening against the surface of osteogenic cells at various stages of differentiation in vitro, as well as evaluating in situ reactivity with human fetal limbs, we isolated three hybridoma cell lines referred to as SB-10, SB-20, and SB-21. Immunocytochemical analyses during osteogenic differentiation demonstrate that SB-10 reacts with MSCs and osteoprogenitors, but no longer reacts with cells once alkaline phosphatase (APase) is expressed. Flow cytometry documents that SB-10 is expressed on the surface of all purified, culture-expanded human MSCs, thus providing further evidence that these cells are a homogeneous population. By contrast, SB-20 and SB-21 do not react with the progenitor cells in situ, but bind to a subset of the APase-positive osteoblasts. None of these antibodies stain terminally differentiated osteocytes in sections of developing bone. Furthermore, these McAbs were not observed to react in samples from chick, rat, rabbit, canine, or bovine bone, although selected extraskeletal human tissues were immunostained. In all cell and tissue specimens examined, SB-20 immunostaining is identical to that observed with SB-21. We have used these McAbs to refine our understanding of the discrete cellular transitions that constitute the osteogenic cell lineage. We suggest a refined model for understanding osteoblast differentiation that is based on the proposition that the sequential acquisition and loss of specific cell surface molecules can be used to define positions of individual cells within the osteogenic cell lineage.
Article
The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.
Article
We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed "periostin." Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription- polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3-E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to betaig-h3, a molecule induced by transforming growth factor beta (TGF-beta) that promotes the adhesion and spreading of fibroblasts. Because TGF-beta has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF-beta increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.
Article
Capillary invasion is a vital regulatory signal during bone morphogenesis that is influenced by angiogenic molecules such as fibroblast growth factor (FGF) and some members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-betas themselves. Bone morphogenetic proteins (BMPs), which are members of the TGF-beta superfamily, have previously not been shown to possess direct angiogenic properties. Osteogenic protein-1 (OP-1; BMP-7) is a potent regulator of cartilage and bone differentiation in vivo. The osteogenic and angiogenic properties of OP-1 at both ortho- and heterotopic sites in adult chacma baboons (Papio ursinus) are enhanced synergistically by the simultaneous application of relatively low doses of TGF-beta1. The single application of relatively high doses of TGF-beta1 (20 ng), and bFGF (500 ng) or relatively low (100 ng) and high (1,000 ng) doses of OP-1 in the chick chorioallantoic membrane (CAM) assay elicited a prominent and (for OP-1) dose-dependent angiogenic response. The binary application of a relatively low dose of OP-1 (100 ng) with a relatively low dose of bFGF (100 ng) or with a relatively low (5 ng) or high (20 ng) dose of TGF-beta1 resulted in a synergistic enhancement of the angiogenic response. The angiogenic effect of the relatively low doses of the combined morphogens was distinctly more pronounced than that of the single application of the relatively high doses of the respective factors. The present findings suggest that these morphogens may be deployed in binary combination in order to accentuate experimental angiogenesis. The cooperative interaction of the different morphogens in the CAM assay may provide important biological clues towards the control of clinical angiogenesis.
Article
The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for alpha-smooth muscle actin (alpha-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for alpha-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, > 68% of PL cells were immunostained for AP; approximately 50% and approximately 51% for OPN and alpha-SMA (p = 0.3), respectively, while only approximately 8% were positively stained for BSP (p < 0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53%, and 56% positive staining for alpha-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A approximately Group B approximately Group C in situ for p > 0.2) except for BSP which was 3 to 4 fold higher in vitro (p < 0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.
Article
It is thought that during development of the periodontium, dental follicle cells, when appropriately triggered, have the ability to differentiate into periodontal ligament fibroblasts, cementoblasts, and osteoblasts. However, the exact mechanisms/factors responsible for initiating cell differentiation are not defined. The purpose of this in vitro study was to further characterize follicle cells and to determine the effects of an enamel matrix-derived protein (EMD) on these cells. Murine follicle cells, transformed with simian virus 40 (SV 40) T antigen-containing virus (SVF cells), were used. SVF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 2% fetal bovine serum (FBS) or 2% FBS plus EMD (100 microg/ml), with and without ascorbic acid (50 microg/ml). For proliferation assays, cells were plated at 500 cells/cm2 in 24-well plates and counted on days 3, 4, and 5. For Northern analysis, total RNA was isolated on days 8, 12, and 18. Induction of mineral nodules by SVF cells was determined by von Kossa staining. EMD had a significant proliferative effect on SVF cells, when compared with 2% FBS control. Based on investigations in situ, follicle cells at the time point used here do not express key mineral-associated markers, e.g., osteocalcin (OCN) or bone sialoprotein (BSP). Significantly, by day 12 in culture, Northern analysis indicated that the follicle cells expressed transcripts for BSP, OCN, and osteopontin (OPN). EMD increased OPN mRNA and decreased OCN mRNA expression. SVF cells were capable of inducing mineralization on day 18, but EMD blocked this activity. These results suggest the follicle cells have the capacity to act as cementoblasts or osteoblasts. Furthermore, EMD can regulate follicle cell activity, thus suggesting that epithelial-mesenchymal interactions may be important during development of periodontal tissues.
Article
Mesenchymal stem cells (MSCs) are pluripotent cells in the bone marrow that have the capacity to differentiate along a number of connective tissue lineages, including cartilage, bone, adipose tissue, and stroma. The SH-3 and SH-4 monoclonal antibodies recognize epitopes present on the surface of human MSCs. This study describes the isolation and characterization of the antigen that is recognized by these antibodies. A protein of molecular weight approximately 67 kDa was immunoprecipitated from a solubilized membrane preparation of human MSCs using the SH-3 antibody. Analysis of peptides derived from this protein by mass spectrometry and sequencing identified it as CD73 (ecto-5'-nucleotidase). The SH-4 antibody was also shown to react with purified bovine CD73 by immunoblotting, but the SH-3 antibody failed to react with the bovine protein. These results indicate that both SH-3 and SH-4 epitopes are present on CD73, but they are distinct. CD73, present in lymphoid tissue, plays a role in the activation of B-lymphocytes and in signal transduction in the hematopoietic compartment of bone marrow. The role that CD73 may play in bone marrow stromal interactions and in the differentiation of MSCs is discussed.
Article
Growth factors and other paracrine signal molecules regulate communication between cells in all developing organs. During tooth morphogenesis, molecules in several conserved signal families mediate interactions both between and within the epithelial and mesenchymal tissue layers. The same molecules are used repeatedly during advancing development, and several growth factors are coexpressed in epithelial signaling centers. The enamel knots are signaling centers that regulate the patterning of teeth and are associated with foldings of the epithelial sheet. Different signaling pathways form networks and are integrated at many levels. Many targets of the growth factors have been identified, and mutations in several genes within the signaling networks cause defective tooth formation in both humans and mice.
Article
When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. However, factors and mechanisms regulating follicle cell differentiation remain undefined. This study focused on determining the ability of bone morphogenetic protein (BMP) 2 to promote the differentiation of follicle cells and periodontal ligament (PDL) cells along a cementoblast/ osteoblast pathway. Follicle cells and PDL cells were isolated from the first molar region of CD-1 mice and immortalized with SV40. Both cell types expressed BMP-4 and BMP receptors (BMPR) IA and II, but only follicle cells expressed BMP-2 mRNA. Cells were exposed to recombinant human BMP (rhBMP)-2 (0-100 ng/ml) and Northern blots were used to determine the expression of mineral-associated markers. BMP-2, in a dose- and time-dependent manner, induced cementoblast/osteoblast differentiation of follicle cells, as reflected by enhanced core binding factor alpha (Cbfal), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineral formation. U0126, a specific inhibitor of MEK-1/2 members of the MAPK family, abolished BMP-2-mediated expression of BSP and OCN. In contrast, exposure of PDL cells to BMP-2 resulted in modest expression of OCN and minimal promotion of mineralization. These results suggest that BMP-2 triggers follicle cells to differentiate toward a cementoblast/osteoblast phenotype and that the MAPK pathway is involved.
Article
To address the molecular mechanisms of cementogenesis, we have isolated dental follicle cells and examined them to see if they contain cementoblast progenitors. Dental follicle tissue was dissected from the root surface of bovine tooth germ and cells were released by digestion with bacterial collagenase. The released cells were maintained as a bovine dental follicle cells (BDFC). To elucidate the differentiation capacity of BDFC, they were transplanted into severe combined immunodeficiency (SCID) mice for 4 weeks. Transplanted BDFC formed cementumlike matrix; in contrast, bovine alveolar osteoblast (BAOB) transplants formed bonelike matrix, and bovine periodontal ligament cells (BPDL) formed a small amount of the cementumlike matrix. Immunohistochemical analysis showed that cementumlike matrix was positive for anti-cementum attachment protein monoclonal antibody, whereas bone-like matrix was negative. These results indicated that the BDFC contained cementoblast progenitors that were able to differentiate to cementoblasts in vivo. They also indicated that the BDFC are phenotypically distinct from BAOB and BPDL, and provide a useful model for investigating molecular mechanisms of cementogenesis.
Article
Periodontal diseases that lead to the destruction of periodontal tissues--including periodontal ligament (PDL), cementum, and bone--are a major cause of tooth loss in adults and are a substantial public-health burden worldwide. PDL is a specialised connective tissue that connects cementum and alveolar bone to maintain and support teeth in situ and preserve tissue homoeostasis. We investigated the notion that human PDL contains stem cells that could be used to regenerate periodontal tissue. PDL tissue was obtained from 25 surgically extracted human third molars and used to isolate PDL stem cells (PDLSCs) by single-colony selection and magnetic activated cell sorting. Immunohistochemical staining, RT-PCR, and northern and western blot analyses were used to identify putative stem-cell markers. Human PDLSCs were transplanted into immunocompromised mice (n=12) and rats (n=6) to assess capacity for tissue regeneration and periodontal repair. Findings PDLSCs expressed the mesenchymal stem-cell markers STRO-1 and CD146/MUC18. Under defined culture conditions, PDLSCs differentiated into cementoblast-like cells, adipocytes, and collagen-forming cells. When transplanted into immunocompromised rodents, PDLSCs showed the capacity to generate a cementum/PDL-like structure and contribute to periodontal tissue repair. Our findings suggest that PDL contains stem cells that have the potential to generate cementum/PDL-like tissue in vivo. Transplantation of these cells, which can be obtained from an easily accessible tissue resource and expanded ex vivo, might hold promise as a therapeutic approach for reconstruction of tissues destroyed by periodontal diseases.
Article
Mesenchymal stem cells (MSCs) are progenitors of all connective tissue cells. In adults of multiple vertebrate species, MSCs have been isolated from bone marrow (BM) and other tissues, expanded in culture, and differentiated into several tissue-forming cells such as bone, cartilage, fat, muscle, tendon, liver, kidney, heart, and even brain cells. Recent advances in the practical end of application of MSCs toward regeneration of a human-shaped articular condyle of the synovial joint is one example of their functionality and versatility. The present review not only outlines several approaches relevant to the isolation and therapeutic use of MSCs, but also presents several examples of phenotypic and functional characterization of isolated MSCs and their progeny.
Article
A cementoblast progenitor cell line designated BCPb8 was successfully isolated from dental follicle cells immortalized with Bmi-1 and hTERT. BCPb8 showed the potential to differentiate into cementoblasts on implantation into immunodeficient mice. BCPb8 was confirmed to be the first established cementoblast progenitor cell line and will provide a useful model for investigating cementogenesis. The dental follicle is the mesenchymal tissue surrounding the developing tooth germ. During tooth root development, progenitor cells present in the dental follicle are believed to play a central role in the formation of periodontal components (cementum, periodontal ligament, and alveolar bone). However, little more is known about the biology of these progenitors. Previously, we observed that cultured bovine dental follicle cells (BDFCs) contained putative cementoblast progenitors. To further analyze the biology of these cells, we attempted to isolate cementoblast progenitors from immortalized BDFC through expression of the polycomb group protein, Bmi-1, and human telomerase reverse transcriptase (hTERT). BDFCs were transduced with replication-deficient retroviruses carrying human Bmi-1(LXSN-Bmi-1), and hTERT (LXSH-hTERT) for immortalization. Single cell clones were established from immortalized BDFC, and differentiation into cementoblasts was assessed by implantation into immunodeficient mice. BDFCs expressing Bmi-1 and hTERT showed an extended life span-90 population doublings more than normal BDFCs-and still contained cells with the potential to differentiate into cementoblasts on implantation into immunodeficient mice. From these cells, we established a clonal cell line, designated BCPb8, which formed cementum-like tissue that was reactive to the anti-cementum-specific monoclonal antibody 3G9 and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin, and type I collagen on implantation. Thus, by using Bmi-1 and hTERT, we succeeded in immortalizing cementoblast progenitor cells from BDFC without affecting differentiation potential. The BCPb8 cell line is the first immortalized clonal cell line of cementoblast progenitors and could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for patients with periodontitis.
Article
Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the transforming growth factor beta (TGFbeta) superfamily. The roles of BMPs in embryonic development and cellular functions in postnatal and adult animals have been extensively studied in recent years. Signal transduction studies have revealed that Smad1, 5 and 8 are the immediate downstream molecules of BMP receptors and play a central role in BMP signal transduction. Studies from transgenic and knockout mice and from animals and humans with naturally occurring mutations in BMPs and related genes have shown that BMP signaling plays critical roles in heart, neural and cartilage development. BMPs also play an important role in postnatal bone formation. BMP activities are regulated at different molecular levels. Preclinical and clinical studies have shown that BMP-2 can be utilized in various therapeutic interventions such as bone defects, non-union fractures, spinal fusion, osteoporosis and root canal surgery. Tissue-specific knockout of a specific BMP ligand, a subtype of BMP receptors or a specific signaling molecule is required to further determine the specific role of a BMP ligand, receptor or signaling molecule in a particular tissue. BMPs are members of the TGFbeta superfamily. The activity of BMPs was first identified in the 1960s (Urist, M.R. (1965) "Bone formation by autoinduction", Science 150, 893-899), but the proteins responsible for bone induction remained unknown until the purification and sequence of bovine BMP-3 (osteogenin) and cloning of human BMP-2 and 4 in the late 1980s (Wozney, J.M. et al. (1988) "Novel regulators of bone formation: molecular clones and activities", Science 242, 1528-1534; Luyten, F.P. et al. (1989) "Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation", J. Biol. Chem. 264, 13377-13380; Wozney, J.M. (1992) "The bone morphogenetic protein family and osteogenesis", Mol. Reprod. Dev. 32, 160-167). To date, around 20 BMP family members have been identified and characterized. BMPs signal through serine/threonine kinase receptors, composed of type I and II subtypes. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig, B.B. et al. (1994) "Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells", Mol. Cell. Biol. 14, 5961-5974; ten Dijke, P. et al. (1994) "Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4", J. Biol. Chem. 269, 16985-16988; Macias-Silva, M. et al. (1998) "Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2", J. Biol. Chem. 273, 25628-25636). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita, H. et al. (1995) "Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects", J. Cell. Biol. 130, 217-226; Rosenzweig, B.L. et al. (1995) "Cloning and characterization of a human type II receptor for bone morphogenetic proteins", Proc. Natl Acad. Sci. USA 92, 7632-7636; Kawabata, M. et al. (1995) "Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor", J. Biol. Chem. 270, 5625-5630). Whereas BMPR-IA, IB and II are specific to BMPs, ActR-IA, II and IIB are also signaling receptors for activins. These receptors are expressed differentially in various tissues. Type I and II BMP receptors are both indispensable for signal transduction. After ligand binding they form a heterotetrameric-activated receptor complex consisting of two pairs of a type I and II receptor complex (Moustakas, A. and C.H. Heldi (2002) "From mono- to oligo-Smads: the heart of the matter in TGFbeta signal transduction" Genes Dev. 16, 67-871). The type I BMP receptor substrates include a protein family, the Smad proteins, that play a central role in relaying the BMP signal from the receptor to target genes in the nucleus. Smad1, 5 and 8 are phosphorylated by BMP receptors in a ligand-dependent manner (Hoodless, P.A. et al. (1996) "MADR1, a MAD-related protein that functions in BMP2 signaling pathways", Cell 85, 489-500; Chen Y. et al. (1997) "Smad8 mediates the signaling of the receptor serine kinase", Proc. Natl Acad. Sci. USA 94, 12938-12943; Nishimura R. et al. (1998) "Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12", J. Biol. Chem. 273, 1872-1879). After release from the receptor, the phosphorylated Smad proteins associate with the related protein Smad4, which acts as a shared partner. This complex translocates into the nucleus and participates in gene transcription with other transcription factors (). A significant advancement about the understanding of in vivo functions of BMP ligands, receptors and signaling molecules has been achieved in recent years. Figure 1 BMP signaling and its regulation. BMP signals are mediated by type I and II BMP receptors and their downstream molecules Smad1, 5 and 8. Phosphorylated Smad1, 5 and 8 proteins form a complex with Smad4 and then are translocated into the nucleus where they interact with other transcription factors, such as Runx2 in osteoblasts. BMP signaling is regulated at different molecular levels: (1) Noggin and other cystine knot-containing BMP antagonists bind with BMP-2, 4 and 7 and block BMP signaling. Over-expression of noggin in mature osteoblasts causes osteoporosis in mice ( Devlin et al., 2003 ; Wu et al., 2003 ). (2) Smad6 binds type I BMP receptor and prevents Smad1, 5 and 8 to be activated ( Imamura et al., 1997 ). Over-expression of Smad6 in chondrocytes causes delays in chondrocyte differentiation and maturation ( Horiki et al., 2004 ). (3) Tob interacts specifically with BMP activated Smad proteins and inhibits BMP signaling. In Tob null mutant mice, BMP signaling is enhanced and bone formation is increased ( Yoshida et al., 2000 ). (4) Smurf1 is a Hect domain E3 ubiquitin ligase. It interacts with Smad1 and 5 and mediates the degradation of these Smad proteins ( Zhu et al., 1999 ). (5) Smurf1 also recognizes bone-specific transcription factor Runx2 and mediates Runx2 degradation ( Zhao et al., 2003 ). (6) Smurf1 also forms a complex with Smad6, is exported from the nucleus and targeted to the type I BMP receptors for their degradation ( Murakami et al., 2003 ). Over-expression of Smurf1 in osteoblasts inhibits postnatal bone formation in mice ( Zhao et al., 2004 ).
Article
The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. It is believed that this tissue contains stem cells and lineage committed progenitor cells or precursor cells (PCs) for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the isolation of PCs derived from dental follicle of human third molar teeth. These fibroblast-like, colony forming and plastic adherent cells expressed putative stem cell markers Notch-1 and Nestin. We compared gene expressions of PCs, human mesenchymal stem cells (hMSCs), periodontal ligament cells (PDL-cells) and osteoblasts (MG63) for delimitation of PCs. Interestingly, PCs expressed higher amounts of insulin-like growth factor-2 (IGF-2) transcripts than hMSCs. Differentiation capacity was demonstrated under in vitro conditions for PCs. Long-term cultures with dexamethasone produced compact calcified nodules or appeared as plain membrane structures of different dimensions consisting of a connective tissue like matrix encapsulated by a mesothelium-like cellular structure. PCs differentially express osteocalcin (OCN) and bone sialoprotein (BS) after transplantation in immunocompromised mice but without any sign of cementum or bone formation. Therefore, our results demonstrate that cultured PCs are unique undifferentiated lineage committed cells residing in the periodontium prior or during tooth eruption.
Article
Identification, characterization, and potential application of mesenchymal stem cells (MSC) derived from human dental tissues. Dental pulp and periodontal ligament were obtained from normal human impacted third molars. The tissues were digested in collagenase/dispase to generate single cell suspensions. Cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 100 microM l-ascorbate-2-phosphate. Magnetic and fluorescence activated cell sorting were employed to characterize the phenotype of freshly isolated and ex vivo expanded cell populations. The developmental potential of cultured cells was assessed following co-transplantation with hydroxyapetite/tricalcium phosphate (HA/TCP) particles into immunocompromised mice for 8 weeks. MSC were identified in adult human dental pulp (dental pulp stem cells, DPSC), human primary teeth (stem cells from human exfoliated deciduous teeth, SHED), and periodontal ligament (periodontal ligament stem cells, PDLSC) by their capacity to generate clongenic cell clusters in culture. Ex vivo expanded DPSC, SHED, and PDLSC populations expressed a heterogeneous assortment of makers associated with MSC, dentin, bone, smooth muscle, neural tissue, and endothelium. PDLSC were also found to express the tendon specific marker, Scleraxis. Xenogeneic transplants containing HA/TCP with either DPSC or SHED generated donor-derived dentin-pulp-like tissues with distinct odontoblast layers lining the mineralized dentin-matrix. In parallel studies, PDLSC generated cementum-like structures associated with PDL-like connective tissue when transplanted with HA/TCP into immunocompromised mice. Collectively, these data revealed the presence of distinct MSC populations associated with dental structures with the potential of stem cells to regenerate living human dental tissues in vivo.
Article
The induction of bone formation starts by erecting scaffolds of smart biomimetic matrices acting as insoluble signals affecting the release of soluble osteogenic molecular signals. The cascade of bone differentiation by induction develops as a mosaic structure singly initiated by the osteogenic proteins of the transforming growth factor-beta (TGF-beta) supergene family. The osteogenic signals when combined with an insoluble signal or substratum initiate de novo bone formation by induction and are deployed singly, synergistically and synchronously to sculpt the architecture of the mineralized bone/bone marrow organ. The osteogenic proteins of the TGF-beta superfamily are the common molecular initiators deployed for embryonic development and the induction of bone in postnatal osteogenesis, whereby molecules exploited in embryonic development are re-deployed in postnatal tissue morphogenesis as a recapitulation of embryonic development. The pleiotropy of the osteogenic proteins of the TGF-beta superfamily is highlighted by the apparent redundancy of molecular signals initiating bone formation by induction including the TGF-beta isoforms per se, powerful inducers of endochondral bone but in the primate only. Bone induction by the TGF-beta isoforms in the primate is site and tissue specific with substantial endochondral bone induction in heterotopic sites but with absent osteoinductivity in orthotopic calvarial sites on day 30 and only limited osteogenesis pericranially on day 90. Ebaf/Lefty-A, a novel member of the TGF-beta superfamily, induces chondrogenesis in calvarial defects of Papio ursinus and bone regeneration across the defect on day 30 and 90, respectively. The strikingly pleiotropic effects of the bone morphogenetic and osteogenic proteins (BMPs/OPs) spring from amino acid sequence variations in the carboxy-terminal domain and in the transduction of distinct signalling pathways by individual Smad proteins after transmembrane serine/threonine kinase complexes of type I and II receptors. Predictable bone regeneration in clinical contexts requires information concerning the expression and cross regulation of gene products of the TGF-beta superfamily. OP-1, BMP-3, TGF-beta1 and type IV collagen mRNAs expression correlates to the morphological induction and maintenance of engineered ossicles by the hOP-1 osteogenic devices in the non-human primate P. ursinus. Amino-acid sequence variations amongst BMPs/OPs in the carboxy terminal domain confer the structure/activity profile responsible for the pleiotropic activity that controls tissue induction and morphogenesis of a variety of tissues and organs by different BMPs/OPs which are helping to engineer skeletal tissue regeneration in molecular terms.
Article
Repair of tooth supporting alveolar bone defects caused by periodontal and peri-implant tissue destruction is a major goal of reconstructive therapy. Oral and craniofacial tissue engineering has been achieved with limited success by the utilization of a variety of approaches such as cell-occlusive barrier membranes, bone substitutes and autogenous block grafting techniques. Signaling molecules such as growth factors have been used to restore lost tooth support because of damage by periodontal disease or trauma. This paper will review emerging periodontal therapies in the areas of materials science, growth factor biology and cell/gene therapy. Several different polymer delivery systems that aid in the targeting of proteins, genes and cells to periodontal and peri-implant defects will be highlighted. Results from preclinical and clinical trials will be reviewed using the topical application of bone morphogenetic proteins (BMP-2 and BMP-7) and platelet-derived growth factor-BB (PDGF) for periodontal and peri-implant regeneration. The paper concludes with recent research on the use of ex vivo and in vivo gene delivery strategies via gene therapy vectors encoding growth promoting and inhibiting molecules (PDGF, BMP, noggin and others) to regenerate periodontal structures including bone, periodontal ligament and cementum.
Article
Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.
Article
We isolated dental papilla from impacted human molar and proliferated adherent fibroblastic cells after collagenase treatment of the papilla. The cells were negative for hematopoietic markers but positive for CD29, CD44, CD90, CD105, and CD166. When the cells were further cultured in the presence of beta-glycerophosphate, ascorbic acid, and dexamethasone for 14 days, mineralized areas together with osteogenic differentiation evidenced by high alkaline phosphatase activity and osteocalcin contents were observed. The differentiation was confirmed at both protein and gene expression levels. The cells can also be cryopreserved and, after thawing, could show in vivo bone-forming capability. These results indicate that mesenchymal type cells localize in dental papilla and that the cells can be culture expanded/utilized for bone tissue engineering.
Article
Stem cells have been used for regenerative therapies in various fields. The proportion of cells that possess stem cell properties in human periodontal ligament (PDL) cells is not yet well understood. In this study, we quantitatively characterized human PDL cells to clarify their stem cell properties, including self-renewal, multipotency, and stem cell marker expression. PDL cells were obtained from extracted premolar or wisdom teeth, following which a proliferation assay for self-renewal, a differentiation assay for multipotency, immunostaining for STRO-1, and fluorescence-activated cell sorter (FACS) analysis for stem cell markers (including CD105, CD166, and STRO-1) were performed. Approximately 30% of 400 PDL cells were found to possess replicative potential and formed single-cell colonies, and 30% of these colonies displayed positive staining for STRO-1, 20% differentiated into adipocytes and 30% differentiated into osteoblasts. FACS analysis revealed that PDL cells, including cell populations, expressed the stem cell markers CD105, CD166, and STRO-1. The findings of this study indicated that PDL cells possess crucial stem cell properties, such as self-renewal and multipotency, and express the mesenchymal stem cell markers CD105, CD166, and STRO-1 on their cell surface, although there were some variations. Thus, PDL cells can be used for periodontal regenerative procedures.