Clinical AnatomyVolume 30, Issue 3 p. 286-287 In Remembrance A Memorial to Donald R. Cahill, Ph.D Gary E. Wise, Corresponding Author Gary E. Wise gewise6318@yahoo.com Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LouisianaCorrespondence to: Gary E. Wise, Professor and Head, Emeritus, Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA. E-mail: gewise6318@yahoo.comSearch for more papers by this author Gary E. Wise, Corresponding Author Gary E. Wise gewise6318@yahoo.com Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LouisianaCorrespondence to: Gary E. Wise, Professor and Head, Emeritus, Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA. E-mail: gewise6318@yahoo.comSearch for more papers by this author First published: 13 February 2017 https://doi.org/10.1002/ca.22841Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume30, Issue3April 2017Pages 286-287 RelatedInformation

Objectives: Primary isolated dental follicle stem cells (DFSCs) possess a strong osteogenesis capability, and such capability is reduced during in vitro culture. Because dentin matrix protein 1 (DMP1) is essential in the maturation of osteoblasts, our objectives were to determine (1) the expression of DMP1 in the DFSCs, (2) the correlation between DMP1 expression and osteogenic capability of DFSCs, and (3) the ability of DMP1 to promote osteogenic differentiation of DFSCs.Methods: DFSCs and their non-stem cell counterpart dental follicle cells (DFC) were established from postnatal rat pups. Expression of DMP1 in the DFSCs and DFC was determined using real-time RT-PCR and western blotting. Different passages of DFSCs were subjected to osteogenic induction. The correlation between osteogenesis and DMP1 expression was analyzed. Then, expression of DMP1 in the DFSCs was knocked-down using siRNA, followed by osteogenic induction to evaluate the effect of DMP1-knockdown. Finally, the late passage DFSCs with reduced DMP1 expression and osteogenic capability were cultured in osteogenic induction medium containing mouse recombinant DMP1 (mrDMP1) to determine if DMP1 can restore osteogenesis of DFSCs.Results: DFSCs expressed much higher levels of DMP1 than did DFC. DMP1 expression was correlated with the osteogenic capability of DFSCs. Knockdown of DMP1 expression markedly decreased the osteogenesis and osteogenic gene expression in the DFSCs whereas adding mrDMP1 protein to the osteogenic induction medium enhanced osteogenesis.Conclusions: DMP1 is highly expressed in the DFSCs, but minimally expressed in non-stem cell DFC. DMP1 appears to play an important role for osteogenic differentiation of the DFSCs. (C) 2014 Elsevier Ltd. All rights reserved.

The dental follicle (DF) plays an essential role in tooth eruption via regulation of bone resorption and bone formation. Bone morphogenetic protein-6 (BMP6) expression in the DF is coincident with bone growth in the tooth crypt. DF stem cells (DFSCs) have been shown to possess strong osteogenic capability. This study aims to determine the expression of BMP6 in DFSCs and to elucidate the role of BMP6 in the osteogenesis of DFSCs. DFSCs and their non-stem cell counterpart, DF cells (DFCs), were obtained from the DFs of rat pups. We showed that expression of BMP6 was significantly higher in the DFSCs than in the DFCs. DFSCs lost osteogenic capability during in vitro expansion, and DFSCs in late passages had reduced BMP6 expression as compared to early passages of DFSCs when they were subjected to osteogenic induction. Addition of exogenous human recombinant BMP6 (hrBMP6) to the osteogenic medium dramatically enhanced the osteogenesis of the late-passage DFSCs. Knockdown of BMP6 by short interfering RNA in the DFSCs in early passages resulted in a decrease in osteogenesis, which could be restored by addition of hrBMP6. We concluded that DFSCs need to express high levels of BMP6 to maintain their osteogenesis capability. Increased BMP6 expression seen in vivo in the DF may reflect the activation of DFSCs for osteogenic differentiation for bone growth during tooth eruption.

Founded 1945 as ‘Acta Anatomica’ by R. Chambers, G. Glimstedt, T. Peterfi and G. Wolf-Heidegger Continued 1962–1974 by E.A. Boyden, 1955–1980 by A. Delmas, 1972–1980 by F. Walberg, 1945–1980 by G. Wolf-Heidegger, 1981–1988 by R. O’Rahilly, Davis, Calif., 1989–1990 by G.E. Goslow, Jr., Providence, R.I., 1981–1992 by W. Lierse, Hamburg, since 1992 by H.-W. Denker, Essen, and A.W. English, Atlanta, Ga., continued 1999 as ‘Cells Tissues Organs’ by H.-W. Denker, Essen, and A.W. English, Atlanta, Ga.

OBJECTIVES:Adult stem cells (ASCs) remain in a slowly cycling/quiescent state under normal physiological conditions, but they can be awakened from this by certain factors, such as injury signals. Previously, our group has shown that dental follicle stem cells (DFSCs) appear to proliferate more rapidly than their non-stem cell counterparts at elevated temperatures. The study described here has aimed to (i) elucidate optimal temperature in which to culture DFSCs, (ii) determine whether elevated temperatures could enhance differentiation capability of DFSCs and (iii) characterize stem cell and osteogenic marker expression of DFSCs at elevated temperatures. MATERIALS AND METHODS:DFSCs obtained from rat first molars were cultured at 37 (control), 38, 39, 40 and 41 ºC. Cell proliferation was evaluated by Alamar blue reduction assay and mean numbers of viable dissociated cells. Osteogenic differentiation was evaluated after 7 or 14 days osteogenic induction. Expression of selected marker genes was also assessed during proliferation and differentiation of the cells. RESULTS:Increased cell proliferation was seen at heat-stress temperatures of 38º, 39º and 40 ºC. DFSCs revealed maximal osteogenesis when cultured at 39 and 40 ºC. Moreover, some stem cell and osteogensis-associated markers had elevated expression in heat-stress conditions. CONCLUSIONS:Under determined heat-stress conditions, DFSCs increased their proliferation, osteogenic differentiation and expression of some marker genes. Thus, it is likely that elevated temperature could serve as a factor to activate adult stem cells.

Tooth eruption requires osteoclastogenesis and subsequent bone resorption. Secreted frizzled-related protein-1 (SFRP-1) negatively regulates osteoclastogenesis. Our previous studies indicated that SFRP-1 is expressed in the rat dental follicle (DF), with reduced expression at days 3 and 9 close to the times for the major and minor bursts of osteoclastogenesis, respectively; but it remains unclear as to what molecules contribute to its reduced expression at these critical times. Thus, it was the aim of this study to determine which molecules regulate the expression of SFRP-1 in the DF. To that end, the DF cells were treated with cytokines that are maximally expressed at days 3 or 9, and SFRP-1 expression was determined. Our study indicated that colony-stimulating factor-1 (CSF-1), a molecule maximally expressed in the DF at day 3, down-regulated SFRP-1 expression. As to endothelial monocyte-activating polypeptide II (EMAP-II), a highly expressed molecule in the DF at day 3, it had no effect on the expression of SFRP-1. However, when EMAP-II was knocked down by siRNA, the expression of SFRP-1 was elevated, and this elevated SFRP-1 expression could be reduced by adding recombinant EMAP-II protein. This suggests that EMAP-II maintained a lower level of SFRP-1 in the DF. TNF-α is a molecule maximally expressed at day 9, and this study indicated that it also down-regulated the expression of SFRP-1 in the DF cells. In conclusion, CSF-1 and EMAP-II may contribute to the reduced SFRP-1 expression seen on day 3, while TNF-α may contribute to the reduced SFRP-1 expression at day 9.

Tooth eruption is a localized event that requires a dental follicle (DF) to regulate the resorption of alveolar bone to form an eruption pathway. During the intra-osseous phase of eruption, the tooth moves through this pathway. The mechanism or motive force that propels the tooth through this pathway is controversial but many studies have shown that alveolar bone growth at the base of the crypt occurs during eruption. To determine if this bone growth (osteogenesis) was causal, experiments were designed in which the expression of an osteogenic gene in the DF, bone morphogenetic protein-6 (Bmp6), was inhibited by injection of the first mandibular molar of the rat with a small interfering RNA (siRNA) targeted against Bmp6. The injection was followed by electroporation to promote uptake of the siRNA. In 45 first molars injected, eruption was either delayed or completely inhibited (seven molars). In the impacted molars, an eruption pathway formed but bone growth at the base of the crypt was greatly reduced compared with the erupted first-molar controls. These studies show that alveolar bone growth at the base of the crypt is required for tooth eruption and that Bmp6 may be essential for promoting this growth.

OBJECTIVES:Isolation and purification of adult stem cells (ASC) are a great challenge. Our objectives were to determine whether ASC are more heat-tolerant than non-stem cells, and to explore if ASC could be enriched by heat-stress treatments. MATERIALS AND METHODS:Rat dental follicle cells were cultured in a variety of media to obtain either a heterogeneous cell population (H-DFC) consisting of stem cells and non-stem cells, or a homogenous cell population (DFC) containing non-stem cells only. Real-time RT-PCR was conducted to compare expression of heat-shock proteins (HSPs) between the two populations. To study heat tolerance, H-DFC and DFC were incubated under heat-stress conditions and cell proliferation was evaluated by alamar blue reduction assay. Furthermore, cells resulting from heat-stress treatments were evaluated for differentiation capability and expression of stem cell markers. RESULTS:H-DFC expressed higher levels of HSP110, HSP70s and HSP27s than did DFC. H-DFC increased levels of proliferation at 40 °C compared to controls grown at 37 °C; no significant reduction in proliferation occurred at temperatures below 40.5 °C. In contrast, DFC showed significant reduction in proliferation under all heat-stress treatments. Heat-stressed H-DFC had increased differentiation capability and increased expression of stem cell markers. CONCLUSION:Stem cells appear to be more tolerant to heat stress than non-stem cells. Incubation of a heterogeneous cell population in heat-stress conditions resulted in increased stem cell numbers.

Adult stem cells (ASC) have been found in many tissues and are of great therapeutic potential due to their capability of differentiation. However, ASC comprise only a small fraction of the tissues. In order to use ASC for therapeutic purposes, it is important to obtain relatively pure stem cells in large quantities. Current methods for stem cell purification are mainly based on marker-dependent cell sorting techniques, which have various technical difficulties. In this study, we have attempted to develop novel conditions to favor the growth of the dental follicle stem cells (DFSC) such that the resultant cell populations are enriched in stem cells. Specifically, a heterogeneous dental follicle cell (H-DFC) population containing stem cells and homogenous non-stem cell dental follicle cell population were cultured at 1% or 5% hypoxic conditions. Only the heterogeneous population could increase proliferation in the hypoxic condition whereas the homogenous DFC did not change their proliferation rate. In addition, when the resultant cells from the heterogonous population were subjected to differentiation, they appeared to have a higher capacity of adipogenesis and osteogenesis as compared to the controls grown in the normal atmosphere (normoxic condition). These hypoxia-treated cells also express higher levels of some stem cell markers. Together, these data suggest that stem cells are enriched by culturing the heterogeneous cell populations in a reduced O(2) condition.

Bone morphogenetic proteins (BMPs) play a role in bone formation. Because BMP‐2 and BMP‐6 are expressed in the dental follicle, we hypothesized that BMPs can stimulate the differentiation of dental follicle stem cells (DFSC) into osteoblasts. Thus, inhibiting BMP signaling could decrease osteogenesis, and in turn delay tooth eruption. To test this, DFSC were cultured in osteogenic induction medium containing BMP‐2, BMP‐6, and LDN (BMP type l receptor inhibitor) in different combinations. After culture for 10 days, calcium deposition detected by Alizarin Red was greater in wells containing both BMP‐2 and BMP‐6, as compared to those containing only BMP‐2 or BMP‐6. No calcium deposition was seen in controls containing only induction medium or in wells containing LDN with or without BMPs. After 14 days of culture, deposition was similar to day 10 except that control wells showed similar deposition to wells of BMP‐2 or BMP‐6 alone. To determine the effect of LDN on eruption, rat pups were injected with LDN from days 7–16 postnatally and examined for eruption of the first mandibular molar (M1). M1 eruption was delayed 1–1.5 days in 80% of the LDN‐treated rats as compared to controls. In conclusion, BMP‐2 or BMP‐6 alone accelerated osteogenesis of DFSC into osteoblasts. The effect of BMP‐2 and BMP‐6 together was synergistic. Inhibition of BMP signaling prevented osteogenic differentiation of DFSC and delayed M1 eruption. Supported by NIH grant 5R01DE008911‐19 to G.E.W and S.Y.

Electroporation DNA transfer is a simple and versatile approach to deliver genes. To develop an optimal electroporation protocol to deliver DNA into cells, we conducted square wave electroporation experiments with using rat dental follicle cells as follows: 1) the cells were electroporated at different electric field strengths with lac Z plasmid; 2) plasmid concentrations were tested to determine the optimal doses; 3) various concentrations of bovine serum albumin or fetal bovine serum were added to the pulsing buffer; and, 4) the pulsing durations were studied to determine the optimal duration. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μ g/ μ l were required to achieve high transfection efficiency. BSA or FBS in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45 to 120 milliseconds (ms) at 375 V/cm. Thus, an improved electroporation protocol was established by optimizing the above parameters. In turn, this electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption.

Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin (IL)-1 and IL-18 toll-like receptor signaling pathways. Because MyD88 is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine expression of the Myd88 gene in vivo in the DFs from the first mandibular molars of postnatal rats from days 1 to 11. The results showed that MyD88 was expressed maximally on day 3. Using small interfering RNA (siRNA) to knock down MyD88 expression in the DF cells also reduced the expression of the nuclear factor-kappa B-1 (NFKB1) and monocyte chemoattractant protein 1 (MCP-1) genes. Interleukin-1 alpha up-regulated the expression of NFKB1, MCP-1, and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL-1 alpha effect. Conditioned medium from DF cells with MyD88 knocked down had reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis, as opposed to controls. In conclusion, the maximal expression of MyD88 in the DF of postnatal day 3 rats may contribute to the major burst of osteoclastogenesis needed for eruption by up-regulating MCP-1 and RANKL expression.

The dental follicle appears to regulate both the alveolar bone resorption and bone formation needed for tooth eruption. Tumor necrosis factor-alpha (TNF-alpha) gene expression is maximally upregulated at postnatal day 9 in the rat dental follicle of the first mandibular molar, a time that correlates with rapid bone growth at the base of the tooth crypt, as well as a minor burst of osteoclastogenesis. TNF-alpha expression is correlated with the expression of bone morphogenetic protein-2 (BMP-2), a molecule expressed in the dental follicle that can promote bone formation. Because BMP-2 signaling may be augmented by bone morphogenetic protein-3 (BMP-3), our objective in this study was to determine if the dental follicle expresses BMP-3 and if TNF-alpha stimulates the dental follicle cells to express BMP-2 and BMP-3. Dental follicles were collected from different postnatal ages of rat pups. Dental follicle cells were incubated with TNF-alpha to study its dosage and time-course effects on gene expression of BMP-2 and BMP-3, as determined by real-time RT-PCR. Next, immunostaining was conducted to confirm if the protein was synthesized and ELISA of the conditioned medium was conducted to determine if BMP-2 was secreted. We found that BMP-3 expression is correlated with the expression of TNF-alpha in the dental follicle and TNF-alpha significantly increased BMP-2 and BMP-3 expression in vitro. Immunostaining and ELISA showed that BMP-2 and BMP-3 were synthesized and secreted. This study suggests that TNF-alpha can upregulate the expression of bone formation genes that may be needed for tooth eruption.

Background Delivery of DNA into the target tissues is an important technique in gene function studies and gene therapy. Surgical treatment of tooth eruption disorders, such as impacted third molars, is a major healthcare cost. Because the dental follicle (DF) is essential for regulating tooth eruption, establishment of local gene transfer protocols is needed to determine the effect of various genes on eruption and to develop gene therapy approaches for inducing the eruption of impacted molars.Methods Plasmids containing lacZ reporter gene were injected into rat mandibles and then electroporated at the designated settings. Mandibles were collected 24 h after electroporation for X-gal staining to evaluate the transfection efficiency. Tissues were collected at various days post-electroporation to determine the expression of the transgene.Results For the DF, depth of injection and pulse number appear to be important. Six pulses can achieve above 80% transfection of the DF at 50 V or 120 V. For alveolar bone (AB) transfection, voltages are important, with 120 V being optimal. Regarding pulse durations, we determined that durations of 20 and 30 ms achieve the maximum transfection in AB and DF, respectively.Conclusions The present study demonstrates for the first time the feasibility of electroporation to locally deliver plasmids into dental tissues. Parameters affecting electroporation to deliver plasmids into the dental tissues were optimized. This protocol could be used to deliver short hairpin RNA or genes of interest into the dental tissues to regulate tooth eruption. Thus, it may be possible to develop nonsurgical treatments for inducing the eruption of impacted teeth. Copyright (c) 2010 John Wiley & Sons, Ltd.

Electroporation is a simple and versatile approach for DNA transfer but needs to be optimized for specific cells. We conducted square wave electroporation experiments for rat dental follicle cells under various conditions. These experiments indicated that the optimal electroporation electric field strength was 375 V/cm, and that plasmid concentrations greater than 0.18 μg/μL were required to achieve high transfection efficiency. BSA or fetal bovine serum in the pulsing buffer significantly improved cell survival and increased the number of transfected cells. The optimal pulsing duration was in the range of 45–120 ms at 375 V/cm. This electroporation protocol can be used to deliver DNA into dental follicle cells to study the roles of candidate genes in regulating tooth eruption. This is the first report showing the transfection of dental follicle cells using electroporation. The parameters determined in this study are likely to be applied to transfection of other fibroblast cells.

OBJECTIVES Tooth eruption requires the presence of a dental follicle (DF), alveolar bone resorption for an eruption pathway, and alveolar bone formation at the base of the bony crypt. The objectives of our investigations have been to determine how the DF regulates both the osteoclastogenesis and osteogenesis needed for eruption. MATERIAL AND METHODS Multiple experimental methods have been employed. RESULTS The DF regulates osteoclastogenesis and osteogenesis by regulating the expression of critical genes in both a chronological and spatial fashion. In the rat 1st mandibular molar there is a major burst of osteoclastogenesis at day 3 postnatally and a minor burst at day 10. At day 3, the DF maximally expresses colony-stimulating factor-1 (CSF-1) to down-regulate the expression of osteoprotegerin (OPG) such that osteoclastogenesis can occur. At day 10, the minor burst of osteoclastogenesis is promoted by upregulation of vascular endothelial growth factor (VEGF) and RANKL in the DF. Spatially, the bone resorption is in the coronal portion of the bony crypt and genes such as RANKL are expressed more in the coronal region of the DF than in its basal one-half. For osteogenesis, bone formation begins at day 3 at the base of the bony crypt and maximal growth is at days 9-14. Osteo-inductive genes such as bone morphogenetic protein-2 (BMP-2) appear to promote this and are expressed more in the basal half of the DF than in the coronal. Conclusion - The osteoclastogenesis and osteogenesis needed for eruption are regulated by differential gene expression in the DF both chronologically and spatially.

The dental follicle (DF) differentiates into the periodontal ligament. In addition, it may be the precursor of other cells of the periodontium, including osteoblasts and cementoblasts. We hypothesized that stem cells may be present in the DF and be capable of differentiating into cells of the periodontium. Stem cells were identified in the DF of the rat first mandibular molar by Hoechst staining, alkaline phosphatase staining, and expression of side-population stem cell markers. These cells were shown to be able to differentiate into osteoblasts/cementoblasts, adipocytes, and neurons. Treating the DF cell population with doxorubicin, followed by incubation in an adipogenesis medium, suggested that the adipocytes originated from stem cells. Thus, a possibly puripotent stem cell population is present in the rat DF.

Teeth move through alveolar bone, whether through the normal process of tooth eruption or by strains generated by orthodontic appliances. Both eruption and orthodontics accomplish this feat through similar fundamental biological processes, osteoclastogenesis and osteogenesis, but there are differences that make their mechanisms unique. A better appreciation of the molecular and cellular events that regulate osteoclastogenesis and osteogenesis in eruption and orthodontics is not only central to our understanding of how these processes occur, but also is needed for ultimate development of the means to control them. Possible future studies in these areas are also discussed, with particular emphasis on translation of fundamental knowledge to improve dental treatments.

Endothelial monocyte-activating polypeptide II (EMAP-II) is an inflammatory cytokine with chemotactic activity. Because the dental follicle (DF) recruits mononuclear cells (osteoclast precursors) to promote the osteoclastogenesis needed for tooth eruption, it was the aim of this study to determine if EMAP-II contributes to this recruitment. Using a DNA microarray, EMAP-II was found to be highly expressed in vivo in the DFs of day 1 to day 11 postnatal rats, with its expression elevated on days 1 and 3. Use of a short interfering RNA (siRNA) to knock down EMAP-II expression resulted in a reduction in the expression of colony-stimulating factor-1 (CSF-1) and monocyte chemoattractant protein-1 (MCP-1) in the DF cells. Addition of EMAP-II protein to the DF cells partially restored the expression of CSF-1 and MCP-1. In chemotaxis assays using either conditioned medium of the DF cells with anti-(EMAP-II) immunoglobulin G added or conditioned medium of DF cells with EMAP-II knocked down by siRNA, migration indexes of bone marrow mononuclear cells were significantly reduced. These results suggest that EMAP-II is another chemotactic molecule in the dental follicle involved in the recruitment of mononuclear cells, and that EMAP-II may exert its chemotactic function directly by recruiting mononuclear cells and indirectly by enhancing the expression of other chemotactic molecules (CSF-1 and MCP-1).