Blockade of TLR2 Inhibits P. gingivalis Suppression of Mineralized Matrix Formation by Human Dental Pulp Stem Cells

Transcription

1 Blockade of TLR2 Inhibits P. gingivalis Suppression of Mineralized Matrix Formation by Human Dental Pulp Stem Cells By: Valerie Tom-Kun Yamagishi A thesis submitted in conformity with the requirements For the degree of Master of Science Faculty of Dentistry University of Toronto Copyright by Valerie Tom-Kun Yamagishi (2011)

2 Blockade of TLR2 Inhibits P. gingivalis Suppression of Mineralized Matrix Formation by Human Dental Pulp Stem Cells Valerie Tom-Kun Yamagishi Master of Science Faculty of Dentistry University of Toronto 2011 Abstract In an effort to re-establish tissue with odontogenic potential in the pulp space of immature permanent teeth, stimulated human dental pulp stem/progenitor cells (hdpscs) have shown potential to differentiate and form mineralized matrix, marked by high expression of dentin sialophosphoprotein (DSPP) and osteocalcin (OCN). Bacterial by-products have been shown to adversely affect cell differentiation. This study investigated the effect of P. gingivalis, a putative endodontic pathogen, and blockage of its host recognition on hdpscs. Stimulated hdpscs were exposed to varying concentrations of P. gingivalis by-product and gene expression of DSPP and OCN was measured. Cells were exposed to TLR2 blocking agents prior to exposure to the byproduct. P. gingivalis affected dose-dependent suppression of the measured gene expression. Blockade of TLR2 inhibited the by-product derived suppression of gene expression. The immune-potential of by-product was confirmed to be detrimental to the differentiation of hdpscs, and this effect could be moderated by TLR2-blockade. ii

3 Acknowledgments I would like to acknowledge the official and unofficial members of my scientific advisory committee, including Drs. Michael Glogauer, (primary principal supervisor), George Huang (secondary principal supervisor), Calvin Torneck, Shimon Friedman, Bernhard Ganss, and Anil Kishen. The numerous hours of their guidance are evident and without them this project would not have been possible. Their mentorship provided me with the perseverance to pursue this project and I hope they recognize to what degree of significance each of their influence has had on me. I would also like to extend gratitude to Dr. Gajanan V. Kulkarni for providing biostatistical consultation. Without the technical support of Siavash Hassanpour, Dr. Jan Kuiper and Dr. Yongqiang Wang, the in-lab trouble-shooting process would have been much more laborious than it seemed at the time. Dr. Clarence Tang was the first person to introduce the idea of studying regenerative endodontics to me. His enthusiasm of the topic encouraged me to continue with this line of research. Most importantly, I would like to thank my dear husband, Dr. Kyle Yamagishi, for the many occasions of driving me to and from the lab at odd hours of the day, eating meals alone, and listening to my roller-coaster journey throughout my project. This study was supported in part by grants from the Canadian Academy of Endodontics Endowment Fund, the Alpha Omega Foundation of Canada, the Dental Research Institute of the University of Toronto, and the National Institutes of Health R01 DE (G.T.-J.H.). iii

4 Table of Contents Abstract _ii Acknowledgements Table of Contents _ iii iv List of Figures and Table _ vi Abbreviations viii Chapter 1: Introduction Regenerative Endodontics Rationale for Regenerative Endodontics Attempts at Pulp Endodontics Obstacles for Pulp Regeneration Characterization of Human Dental Pulp Stem Cells _ Dentin sialophosphoprotein Osteocalcin LPS derived from Porphyromonas gingivalis P. gingivalis and Endodontic Infections _ Atypical Structure and Host Recognition Toll-Like Receptor (TLR) TLR2 Pathway Neutralizing TLR2 Antibody _ SC514, a Pharmacological Inhibitor of IKK _ Statement of the Problem 12 Chapter 2: Materials and Methods _ Cell culture _ 15 iv

5 2.2 Stimulation and exposure to LPS Blockade of TLR2 pathway RNA extraction and cdna preparation _ Real-time quantitative PCR (RT-qPCR) Data analysis 17 Chapter 3: Results _ Characterization of isolated hdpscs _ Effect of P. gingivalis on DSPP and OCN gene expression Effect of TLR2 blockade on suppression of DSPP and OCN gene expression 19 Chapter 4: Discussion _ 20 Chapter 5: Future Directions 26 Figure and Table Legend 27 Figures and Table 30 Chapter 6: Conclusions _41 References 42 v

6 List of Figures and Table Figure 1. A. Molecular structure of pharmacological inhibitor SC514 (Cayman Chemical, Ann Arbor MI). B. One of the reported intracellular pathways downstream of Toll-like Receptor 2 activation. Figure 2. Bioanalyzer electropherogram of total RNA. A. Unstimulated hdpscs B. Stimulated hdpscs C. Stimulated hdpscs exposed to LPS Figure 3. MFOLD analysis of the amplicons for potential secondary structures. A. hgapdh B. hdspp C. hocn Figure 4. Gel analysis of PCR products. A. hgapdh 97 bps B. hdspp 277 bps C. hocn 155 bps Figure 5. Melt Curve for real-time quantitative PCR results. A. hgapdh has a single peak at 82.5 C B. hocn has single peak at 87.5 C Figure 6. hdpscs grown and analyzed in cultures. A. Typical clonogenic hdpscs forming a colony in the culture dish after being isolated from human pulp tissues using enzyme digestion. vi

7 B. hdpscs produced mineralized particles were stained red by Alizarin Red S. Figure 7. Effect of P. gingivalis by-product on DSPP and OCN gene expressions. A. Quantitative real-time PCR was used to detect mrna expression levels of DSPP B. Quantitative real-time PCR was used to detect mrna expression levels of OCN Figure 8. Effect of Anti-TLR2 on P. gingivalis suppression of DSPP and OCN. A. Quantitative real-time PCR was used to detect mrna expression levels of DSPP B. Quantitative real-time PCR was used to detect mrna expression levels of OCN Figure 9. Effect of pharmacologic inhibitor, SC514 on P. gingivalis suppression of DSPP and OCN. A. Quantitative real-time PCR was used to detect mrna expression levels of DSPP B. Quantitative real-time PCR was used to detect mrna expression levels of OCN Table 1. Primer sequences for the genes analysed. vii

8 Abbreviations ABC ANOVA CD DMSO DNA DGP DPP DSP DSPP GAPDH hdpsc HEPES IKK IL-1β IL-1R IRAK LPS MAL α-mem MF MSC MTA NEMO NF-κB OCN OD OPC PAMP PCR Apical bud cells Analysis of variance Cluster of differentiation Dimethyl sulfoxide Deoxyribonucleic acid Dentin glycoprotein Dentin phosphoprotein Dentin sialoprotein Dentin sialophosphoprotein Glyceraldehyde 3-phosphate dehydrogenase Human dental pulp stem/precursor cell 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid Inhibitor of NF-κB kinase Interleukin-1β Interleukin 1 receptor IL-1 receptor-associated kinase Lipopolysaccharide MyD88-adaptor-like α-minimum essential medium Molecular formula Mesenchymal stem cell Mineral trioxide aggregate NF-κB essential modulator Nuclear factor kappa-light-chain-enhancer of activated B cells Osteocalcin Odontoblast Odontoblast progenitor cell Pathogen-related molecular patterns Polymerase chain reaction viii

9 PDGF PLG PLLA RNA RT-PCR RT-qPCR Runx2 SCAP SHED TIR TLR TNF TRAF VEGF Platelet-derived growth factor Polylactic co-glycolic acid Poly-L-lactic acid Ribonucleic acid Reverse transcriptase PCR Real-time quantitative PCR Runt-related transcription factor Stem cells from the apical papilla Stem cells from exfoliated deciduous teeth TLR/IL-1R Toll-like Receptor Tumor necrosis factor TNF receptor-associated factor Vascular endothelial growth factor ix

10 Chapter 1 Introduction 1.1 Regenerative Endodontics Rationale for Regenerative Endodontics Teeth develop in response to ectodermal and ectomesenchymal interaction. During development, signals from ectodermally derived ameloblasts and root sheath epithelium promote differentiation of the ecto-mesenchymally derived papilla cells into the odontoblasts that form dentin. Primary dentinogenesis proceeds at a relatively rapid rate and continues until the root is formed and the pulp space defined. Secondary dentinogenesis, dentin formed after root development is complete, is deposited more slowly throughout life in a generalized but altered pattern than that of primary dentin. Reactionary dentin and reparative dentin formation, in response to microbial stimuli and physical pulp injury, occurs at a rate similar to primary dentin but only at localized sites. Odontoblasts responsible for the dentin formation are end cells and therefore incapable of cell division. New odontoblasts however, can develop from stem cells and progenitor cells in the dental pulp, when required to support dentin maintenance. The outcome for fully formed teeth with irreversible pulp injury to remain functional for long periods of time is deemed excellent where endodontic treatment is successfully undertaken (1). However, the outcome for partially formed teeth experiencing the same injury and treated in a similar manner is significantly poorer (2). This is due to the susceptibility of these immature roots to fracture (2). Autogenous tooth replacement has been attempted as optional treatment in such situations; however a suitable donor tooth is not always available. Research is being undertaken at laboratories throughout the world to develop a customized de novo biological tooth replacement, but the clinical application of this type of tooth replacement is still several years ahead (3-7). More encouraging are current attempts to re-establish in the root canal of partially developed teeth, a functioning pulp or tissue with pulp-like potential, which would be capable of restoring dentinogenesis and tooth formation. 1

11 Pulpal infection and inflammation is compartmentalized until the entire pulp undergoes necrosis (8,9). Before the necrosis is complete, the remaining vital tissue may be revived and may also be stimulated to regenerate the portion that was lost to disease (10,11). This type of regeneration may be augmented by the placement of engineered pulp tissue into the pulp space, and acceleration of the process of dentinogenesis. Both complete and partial pulp regeneration are procedures referred to as regenerative endodontics (12). In order for pulp regeneration to be clinically successful several challenges have to be overcome. Dental pulp is normally a highly vascular tissue that receives its blood supply from limited sites. The most important of these is the apical foramen or foramina where the principal vessels enter and exit (13). Less important, but assuming the role of a co-lateral supply are the lateral canals which are found in different sites along the root and occasionally in the furcation area of multirooted teeth (13). These sites allow sufficient blood flow to maintain the pulp during function and to respond to minor injury. However due to their restricted size and diverse location, it is impossible to re-establish the vasculature once it has been lost to disease. The anatomy of the pulp space in partially developed teeth and the potential for fracture of the roots due to the thinness of the dentin walls are the reason that regenerative procedures have been advocated. The success of such procedures is enhanced by the fact that in partially developed teeth the apical foramen is appreciably larger than that of a fully developed tooth and consequently more amenable to the re-establishment of a new vasculature in support of a new pulp (10). The need to provide adequate space for the re-establishment has been recognized for many years. A pioneer amongst those who have attempted to make this concept clinically feasible was Nygaard- Östby (14) who not only advocated revascularization for developing teeth, but also for developed teeth undergoing endodontic treatment. In these cases, he advised physically enlarging the apical foramen with endodontic files and initiating bleeding into the root canal to promote angiogenesis and apical pulp regeneration. Attempts to re-establish a vasculature in the pulp was reported in 1965 by Ohman, who extracted and then replanted partially developed teeth and reported reestablishment of a vasculature in the middle and apical third of the root canal (15). While not an engineered and predictable event in these cases, they did indicate that revascularization was possible and therefore a goal to pursue in regenerative endodontics. 2

12 The goal of pulp regeneration is to create a tissue that is functionally competent, self-sustaining, and capable of forming dentin. Pulp regeneration began to move forward in the late 1990s when tissue-engineering technology began to show promise (16-20). It was shown that isolated pulp cells could be stimulated to differentiate into odontoblast-like cells and produce a calcified extracellular matrix in vitro (21). Gronthos et al (22) reported on the ability of pulp cells to generate dentin in vivo, and also demonstrated that human pulp/dentin complex could be formed ectopically in immunocompromised mice (22). An obstacle to the clinical application of regenerative endodontics is the role played by bacteria and their by-products. Bacterial invasion of the pulp space is the principal cause of irreversible pulp injury (23) and their colonization of the pulp space makes it difficult for new tissue to become established there in their presence. Modern endodontic treatment regimens are successful in reducing bacteria in root canals by up to 90-99% but they are less effective in eliminating bacterial lipopolysaccharide (LPS), produced by Gram-negative microorganisms (24). Clearly, for regenerative endodontics to be clinically successful methods must be developed to maximize bacterial control, while minimizing co-lateral injury. Furthermore, methods have to be developed that effectively seal the coronal portion of the root canal system to minimize the risk of bacterial ingress and re-colonization of the root canal after regeneration of the pulp has been realized. Finally, cells selected to repopulate the pulp space have to be not only hardy, but also retain a potential to multiply and differentiate into odontoblasts or odontoblast-like cells that are capable of secreting a dentin-like matrix on the existing dentin walls that can be mineralized in order to increase its fracture resistance. These cells are expected to assume the role of odontoblasts and continue to maintain the dentin over time. Implanted cells should also possess a potential to differentiate into endothelial cells and participate in the process of angiogenesis since the future activities of the implanted cells will be dependent upon the presence of an active and responsive vasculature (12). Ideally, a newly formed pulp should also include a sensory component and immuno-competent cells other than odontoblasts; however, how and when these cells would repopulate the pulp and the nature of the role they will play has yet to be determined Attempts at Pulp Regeneration 3

13 Regeneration of a tissue or organ has been the goal of all medical disciplines. Blood clots have been used as a scaffold that provides a rich source of growth factors to aid in tissue repair. In endodontics, Nygaard-Östby in the 1960s (14) and later Myers et al (25) used this method. Subsequent histological studies however indicated that the tissue regenerated in such cases had more of the characteristics of periodontal tissue than pulp tissue (26,27). In 2003, Shi et al (28) reported on the possibility of using stem cells for the regeneration of pulp and dentin. Post-natal dental pulp stem cells (DPSCs) were found to share similar characteristics of mesenchymal stem cells (MSCs) and to form ectopic human pulp-/dentin-like complex, and a dentin-like tissue on the existing human dentin surface in immunocompromised mice (22,29,30). In addition to DPSCs, other stem/progenitor cells from dental tissues have been isolated and characterized, including stem cells from exfoliated deciduous teeth (SHED) (31), periodontal ligament stem cells (hpdlsc) (32), stem cells from the apical papilla (SCAP) (6) and dental follicle progenitor cells (33). These post-natal stem/progenitor cell populations have been shown to possess qualities similar to MSCs, including the ability for self-renewal and multilineage differentiation potential (10). All of the different stem cells considered for endodontic applications have to be characterized to determine their suitability for use in regenerating tissues including pulp and dentin (34,35) Obstacles for Pulp Regeneration Ideally, regenerated pulp tissue should be well vascularized, have similar cell content, density, and architecture as normal pulp, and be capable of giving rise to new odontoblasts-like cells, produce new dentin, and be innervated. Vascularization may be difficult in mature teeth due to the restricted anatomy of the root and pulp space (12). Immature teeth with open apices are better-suited candidates for pulp tissue regeneration, as there is a greater chance for the reestablishment of a blood supply. The use of angiogenic-inducing factors, such as VEGF and/or PDGF, is thought to enhance and accelerate the angiogenesis (12). Synthetic scaffolds, such as PLG, can be fabricated with impregnated growth factors (36-41). Alternatively, the maturation of engineered pulp tissue may have to be carried through multiple stages (42). 4

14 If a good blood supply is achievable optimal cell density and the deposition of high- quality extracellular matrix should occur. In previous studies (43,44), new odontoblast-like cells were reported to form against an existing dentin that has been chemically disinfected, although the physical and chemical properties of the new mineral tissue were not determined. In terms of innervation, regenerated pulp was found to contain ingrown nerve fibers from adjacent neural tissues (12). DPSCs have been shown to produce neurotrophic factors and possess neural differentiation potential (29,41); however, the development of a normal innervation pattern for regenerated pulp poses a considerable challenge. 1.2 Characterization of Human Dental Pulp Stem/Progenitor Cells The site where MSCs reside in an adult is still largely unknown. It has been proposed that they reside in the perivascular areas (28,45). Using stem cell markers such as STRO-1, CD146 and pericyte-associated antigen (3G5), DPSCs were found to be localized in the perivascular and perineural sheath regions (28). The STRO-1 positive region in the pulp of deciduous teeth was found to be similar to that of permanent teeth (i.e. in the perivascular region). STRO-1 staining of apical papilla has shown that the positive stain is located in several sites as well as in the perivascular region (6). STRO-1/CD146/CD44 staining of the periodontal ligament has shown that it can be found in the paravascular and extravascular region (46). Dental stem/progenitor cells are considered to be MSC-like cells, therefore markers that have been used for identifying MSCs have also been used for dental stem cells. Positive markers are: STRO-1, CD13, CD44, CD24, CD29, CD73, CD90, CD105, CD106, CD146, Oct4, Nanog, β2 integrin. Negative markers are: CD14, CD34, CD45 and HLA-DR (28,47-52). Dental stem/progenitor cells have the potential to differentiate along three unique cell lineages: osteo/odontogenic, adipogenic and neurogenic (31,53-55). Differences have been noted between the dental stem/progenitor cell populations and bone marrow-derived MSCs. Dental stem/progenitor cells appear to be more committed to odontogenic as opposed to osteogenic development (22). Subpopulations of DPSCs and SHED appear to have osteogenic, chondrogenic, angiogenic (56), adipogenic and neurogenic potentials (31,53-55). 5

15 DPSCs, SHED and SCAP are all suitable cell sources for pulp/dentin regeneration as they originate from precursor of pulp. DPSCs and SCAP have been shown to produce a pulp-dentin complex when ectopically placed into immunocompromised mice (6,22,29). SHED was shown to form mineralized tissue without the distinct morphology of the pulp-dentin complex (31). Yu et al (57) compared the odontogenic capability of bone marrow mesenchymal stromal cells and DPSCs by co-culturing these cells with apical bud cells (ABCs) in a rat model. They determined that recombined DPSCs/ABCs underwent amelogenesis and dentinogenesis. Bone marrow mesenchymal stromal cell recombinants only developed atypical dentin-pulp complexes without enamel formation (57). These results indicate that DPSCs, SCAP and SHED form pulp and dentin along a more direct route than do bone marrow stem cells. Cordeiro et al (44) used a 1-mm thick horizontal human tooth slice, which was then cast and filled with a biosynthetic scaffold, poly-l-lactic acid (PLLA) to investigate stem cell differentiation. SHED were seeded onto the slices and transplanted into the subcutaneous space of immunocompromised mice. After 2-4 weeks, well-vascularized pulp-like tissue could be seen in the pulp chamber. In addition, odontoblast-like cells expressing dentin sialoprotein (DSP), an odontoblast-specific gene, were localized against the existing dentin surface. No new dentin matrix was formed on the existing dentin surface. Using DPSCs and a collage scaffold that included a grown factor, dentin matrix protein-1, Prescott et al (58) reported that 6 weeks after transplantation into immunocompromised mice, pulp-like tissue could be seen. The collagen scaffold contracted resulting in less optimal results than when PLLA was used. Histological examination revealed no odontoblast-like cells formed against the dentin surface. Huang et al (43) performed a similar in vivo study using a toothfragment. Canals were emptied, enlarged and sealed with MTA cement at one end. Collagen gels containing DPSCs were placed into the canal space and the tooth fragment implanted into the subcutaneous space of severe combined immunodeficiency mice. They found that collagen/cell constructs contracted and failed to fill into the deeper part of canal space and that there was an absence of odontoblast-like cells on the dentin wall. When PLG was used as a scaffold, the pulp canal space was filled with a new well-vascularized pulp-like tissue within 4 months and a layer of a mineralized dentin-like tissue had been deposited onto the dentin surface. 6

16 1.2.1 Dentin Sialophosphoprotein Dentin makes up the major component of mineralized dental tissue (59) and is produced by odontoblasts (60). DSPP was first described in 1997 (61), but its cleaved fragments were discovered much earlier. DSPP is predominantly expressed in dentin and moderately or slightly expressed in other tissues (62,63), suggesting that the transcription of DSPP is highly regulated. Human and mouse genetic studies have shown that DSPP is critical for the mineralization of dentin, but the molecular pathways governing the expression of this gene are poorly understood (64). It has been speculated that DSPP is a multidomain protein with hundreds of posttranslational modifications (65). Porcine DSPP is processed by proteases into three protein products: DSP, dentin glycoprotein (DGP) and DPP (59,66-68). Recently, Sun et al (69) revealed the presence of full-length DSPP and its processed fragments in extracts of pulp/odontoblast and dentin. DPP was first identified in 1967 by Veis and Perry (66) and DSP by Butler et al in 1981(70). Odontoblasts secrete tooth-specific proteins, such as dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) encoded by a single gene called the dentin sialophosphoprotein (DSPP) gene (61,71). DPP is ~90kDa protein and is the most abundant noncollagenous protein in dentin extracellular matrix (72). The unusual feature of DPP is the presence of a large amount of aspartic acid and serine residues. The majority of the serine residues are phosphorylated. High levels of the amino acids produce a polyanionic macromolecule. It is thought that this allows DPP to function in the nucleation and modulation of apatite crystal formation as they could propagate the calcium molecules in a highly oriented manner (64). DSP is ~95kDa glycoprotein, containing little or no phosphate and found in a lesser quantity than DPP (64). DSP is deposited in the predentin and DPP is secreted at the mineralization front and retained in the mineralized dentin (64). Its function is presently undefined. It has little or no effect on in vitro mineralization and does not promote cell attachment (73). A recent in vivo study indicates that DSP may be involved in the initiation of dentin mineralization but not in the maturation of the tissue (74). 7

17 Huang et al (75) reported on the differential expression patterns of DSP and DPP. DPP was mainly present in the inorganic phase and the DSP in the predentin/odontoblast processes. Immunolocalization studies indicated that DSP is more abundant in the predentin than the mineralized dentin (76,77). In the mineralized dentin, DSP appears to be localized in the vicinity of the odontoblast processes along the dentinal tubules (77,78) Osteocalcin The majority of dentin extracellular matrix proteins are common to both dentin and bone. These proteins include type I, III, and V collagens, bone sialoprotein, osteopontin, dentin matrix protein-1, osteonectin and osteocalcin (OCN). A common regulatory pathway between bone and dentin is expected to control such genes. Osteocalcin was reported present in human odontoblasts, detected throughout the length of the odontoblast processes, and located within the enamel matrix (79). Osteocalcin appears in the enamel when the maturation stage is reached (80-82). Osteocalcin was also found to be synthesized by murine odontoblasts and deposited into mineralizing dentin (80-82). 1.3 LPS derived from Porphyromonas gingivalis P. gingivalis and Endodontic Infections Porphyromonas gingivalis (formerly Bacteroides gingivalis) is a black-pigmented, asaccharolytic, obligately anaerobic, non-spore-forming, nonmotile gram-negative rod or coccobacillus. Although occasionally found in sites remote from the oral environment, it has been primarily isolated from the mouth, specifically subgingival plaque, in the tonsils and on the dorsal side of the tongue. P. gingivalis is a major pathogen associated with periodontal diseases (83,84) and in association with Tannerella forsythia and Treponema denticola, it forms the so called Red Complex, implicated in severe marginal periodontitis (85). P. gingivalis has also been reported in endodontic infections using culture-dependent and culture-independent approaches (86-99). Reports on the prevalence of P. gingivalis in primary endodontic infection has varied between 4-29% using cultivation methods (86-92) and in 9-72% 8

18 using culture-independent molecular methods (93-99). The culture-independent studies have strengthened the association between P. gingivalis and primary apical periodontitis Atypical Structure and Host Recognition P. gingivalis has surface components including lipopolysaccharide (LPS), lipoproteins, and fimbriae, which interact with host-expressed toll-like receptors (100,101), that are responsible for initiating the innate immune response when in contact with host tissue (102). Boivin et al (103) was the first reporting on endotoxin extraction from Salmonella typhimurium. Subsequent chemical and biological studies have led to the extraction of endotoxic LPS by Westphal et al. in 1952 (104) using a hot phenol and water method. This paved the way to investigations into the relationship between LPS and the host defense mechanisms. LPS is a key inflammatory mediator and has the ability to potently activate host inflammatory and innate defense responses. It has been proposed that it functions as an important molecule to alert the host of potential bacterial attack. LPS s are large molecules consisting of three parts, the O antigen (or O polysaccharide), the core oligosaccharide, and the lipid A. The O antigen is a repetitive glycan polymer that is exposed on the very outer surface of the bacterial cells and, as a consequence, is a target for recognition by the host. The core oligosaccharide is the component that attaches directly to the lipid A. The lipid A is a phosphorylated glucosamine disaccharide with multiple fatty acids. These hydrophobic fatty acid chains anchor the LPS into the bacterial membrane, and the rest of the LPS projects from the cell surface. The lipid A domain is largely responsible for the toxicity of Gram-negative bacteria. When bacterial cells are lysed by the immune system, fragments of membrane containing lipid A are released into the circulation, propagating the immune response. Although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. The LPS of P. gingivalis has an active center termed the lipid A molecule, which is known to be an active participant in endotoxic activation. Ogawa et al (105) were the first to reveal the chemical structure of P. gingivalis lipid A. The backbone of P. gingivalis lipid A consists of a β-1(1-6)-linked glucosamine disaccharide, which is phosphorylated at the 1-position of the reducing sugar, but 9

19 this lipid A structure lacks an ester-linked phosphate groups which is bound to the hydroxy group at the 4 -position o the nonreducing sugar. Because of its unique lipid A moieties, it was thought that the host recognition of P. gingivalis LPS differed from that of most other Gramnegative bacteria. An in vivo report suggested that TLR2 was specifically required for the host response to P. gingivalis challenge (106). Kocgozlu et al (107) reported that P. gingivalis could activate TLR2 on human epithelial cells. There is still controversy as to whether or not host recognition of P. gingivalis LPS is mediated by TLR2. It was suggested that heterogeneity and contamination of the isolated P. gingivalis lipid A by lipoprotein may have led to the activation of TLR2 (108), which led to the conflicting results. 1.4 Toll-Like Receptor (TLR) TLR2 Pathway TLRs are a class of transmembrane protein receptors present in many types of host cells. They often work together with IL-1 receptors to form a superfamily of receptors that have a common Toll-IL-1 (TIR) domain. There are 3 groups of TIRs: (1) receptors for interleukins produced by antigen processing cells (APCs), (2) receptors for conserved molecular patterns (PAMPs) and (3) adaptor proteins, intracellular proteins that mediate signal initiated by TIR surface domains. Activation of surface domains may be direct or indirect. Their activation by LPS, a surface molecule of Gram-negative microorganisms, is indirect and necessitates linkage of an LPS fragment with an LPS binding protein and the protein MD-2 before activation of the receptor can occur (109). Recognition of LPS can be enhanced by CD14, a surface and soluble protein associated with APCs. Since first being described in the fruit fly Drosophila melanogaster (110), toll-like receptors (TLRs) have proven to be of great interest to those interested in the molecular basis of inflammation. Extensive analysis of TLRs has revealed specificity in terms of ligand recognition, expression in different cell types and tissues, and importantly a role for TLRs in the pathogenesis of multiple diseases involving both the innate and adaptive immune system (110). 10

20 They recognize pathogen-derived factors and also products of inflamed tissue and trigger signaling pathways and protein kinases downstream, most notably the Il-1 receptor-associated kinase (IRAK) family that leads to activation of transcription factors such as nuclear factor-κb (111). This activated pathway leads to activation of the respective transcription factors nuclear factor-κb (NFκB), which in turn induces various immune and inflammatory genes (111). TLR4, that recognizes LPS of most Gram-negative microorganisms, function as a dimer. Difficulty in purifying P. gingivalis LPS results in its recognition by host TLR2. This is likely due to lipoprotein contaminations (108). TLR2 functions as a heterodimer with TLR1 or TLR6 depending upon ligand specificity. TLR2 and TLR4 activate the same intracellular transduction pathway (111); hence binding to either TLR triggers an innate immune response. Currently the ability to manipulate the TIR domain and/or its transduction pathway has led to the development of new and innovative ways of managing clinical infections (111). The TLR signaling pathways depend on cytoplasmic TLR/IL-1R (TIR) domain interactions, which are conserved among all TLRs. Interactions between the TLR TIR domain and the TIR domains of intracellular adaptor proteins initiate the signaling cascade that ultimately leads to transcription factor activation (115). MyD88 is the main adaptor protein implicated in TLR signaling and is considered essential for the induction of inflammatory cytokines triggered by most TLRs. TLR2 activates only MyD88-dependent signaling and requires the MyD88-adaptorlike (MAL) protein to bridge MyD88 to the binding domain of TLR2 ( ). MAL also contains the binding domain for the downstream kinase tumor necrosis factor (TNF) receptorassociated factor 6 (TRAF6), which leads to TRAF6 phosphorylation and NF-κB activation (111). This leads to activation of the NFκB essential modulator (NEMO) which is comprised of three subunits: inhibitor of NF-κB (IκB) kinase (IKK)-α, IKK-γ, and IKK-β, and the subsequent phosphorylation and degradation of the IκB, rendering NF-κB free to translocate from the cytosol to the nucleus and activate κb-dependent genes the pro-inflammatory cytokines (111). Advances in the understanding of signaling pathways activated by TLRs, structural insights into TLRs bound to their ligands and antagonists, and approaches to inhibit TLRs (including antibodies, peptides, and small molecules) are providing possible means by which to interfere with TLR function clinically (111). 11

21 1.4.2 Neutralizing TLR2 Antibody One approach to blocking the TLR2 pathway is to use a neutralizing antibody. Smith et al (120) reported that intravenous anti-tlr4 antibodies significantly reduced the inflammatory responses to LPS derived from Escherichia coli in the lung of rabbits. A similar approach has been reported by Meng et al (121), who used antibodies specific to the extracellular domain of TLR2 in mice was used to prevent sepsis induced by TLR2 ligands. Systemic application of T2.5 upon lipopeptide derived from Bacillus subtilis inhibited the release of inflammatory mediators such as TNF-α and prevented lethal shock-like syndrome. The results indicated that epitope-specific binding of exogenous ligands precedes specific TLR signaling (121) SC514, a Pharmacological Inhibitor of IKK The inhibitor of NF-κB (IκB) kinase (IKK) is the converging point for the activation of the NFκB and is thus a novel target for therapeutic intervention (122). SC514 is a small molecule that is a selective, reversible inhibitor of IKK. SC514 has been shown to inhibit transcription NFκB-dependent genes in IL-1β-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner (122). In addition, Gomez et al (123) demonstrated that pretreatment of rat aortic smooth muscle cells with SC514 significantly reduced inducible nitric oxide synthase and NF-κB DNA binding. 1.5 Statement of the Problem In recent years, there has been a concerted effort to develop therapeutic methods that can regenerate a tissue with odontogenic potential in the root canals of immature permanent teeth that succumbed to pulp necrosis and infection. This development has been driven by the recognized susceptibility of immature roots to fracture when treated with conventional treatment methods (2). To regenerate tissues with odontogenic potential, researchers have described the use of stem cells derived from the pulps of exfoliated deciduous teeth (SHED), the pulps of human permanent teeth (hdpscs), and tissue from the apical papilla of human developing permanent teeth (SCAP) (10,124). In essence, these sources provide a mixed population of pulp cells in which, stem cells and odontoblast progenitor cells (OPCs) may differentiate to form a 12

22 dentin-like mineralized matrix when properly stimulated (125,126). This differentiation is marked by a high gene expression of hard tissue-forming proteins such as dentin sialophosphoprotein (DSPP) (127,128) and osteocalcin (OCN) (129,130). These proteins play an essential role in dentinogenesis and are present in the dentin extracellular matrix that is formed (131). Recent in vivo demonstration of the de novo synthesis of pulp-like tissue forming a continuous layer of dentin-like tissue on the existing dentin of a pulp space by SCAP and DPSCs further highlights the potential in dental tissue regeneration (132). To maximize this potential in infected teeth, it is necessary to neutralized the effect of microorganisms and their by-products that persist after disinfection. A particular concern is the presence of lipopolysaccharide (LPS), a toxic and immuno-stimulatory surface molecule of Gram-negative microorganisms, which can persist in dentin (24,133). LPS derived from Porphyromonas gingivalis, a Gram-negative obligate anaerobe is prevalent (48%) in primary endodontic infections (134) and has been shown to adversely affect DNA production in hdpscs in a dose-dependent manner (135). LPS derived from another putative endodontic pathogen, P. endodontalis, has been shown to up-regulate the production of IL-1β, a pro-inflammatory cytokine (136) that impedes the survival of several mammalian cell lines ( ). Pro-inflammatory cytokines are produced in response to the recognition of pathogen-related molecular patterns (PAMPs), such as an LPS fragment, by a class of trans-membrane receptors know as toll-like receptors (TLRs) located in the cell wall of specific host cells (140). Odontoblasts (OD) and OPCs express TLRs and as a result, retain an ability to participate in the innate immune response (141). Different classes of host TLRs recognize and mediate LPS derived from different microorganisms. LPS derived from enteric bacteria is mediated solely by TLR4 (106), whereas by-products derived from P. gingivalis can be mediated by TLR2 (142). Blockade of TLR4s by anti-tlr4 antibodies has been shown to inhibit pro-inflammatory cytokine production in response to E. coli LPS in rabbit whole blood cell culture (120). This suggests the possibility that blockade of TLR2s may have a similar effect on odontoblast progenitor cells exposed to P. gingivalis by-product, and possibly mitigate the deleterious effects associated with its presence. 13

23 The current study was designed to investigate the effects of intra- and extracellular TLR2 blockade on differentiation and maturation of hdpscs exposed to P. gingivalis by-product as assessed by their DSPP and OCN gene expression and bring attention to the challenges that must be overcome in regenerative endodontics. 14

24 Chapter 2 Materials and Methods 2.1 Cell culture Human DPSCs were isolated based upon methods described in previous reports (43,132). Briefly, teeth and/or pulp tissue was collected from teeth of healthy patients at the University of Maryland Dental Clinics and stored in serum-free culture medium for transportation to the lab for processing. Sample collection conformed to the approved protocols by the Medical Institutional Review Boards at the University of Maryland (Dr. George Huang previous work location). Pulps were minced into 2 x 2 x 1 mm fragments and digested in a solution of 3 mg/ml type I collagenase and 4 mg/ml dispase for 30 to 60 minutes at 37 C (Sigma, St. Louis, MO). Cell suspensions were obtained by passing the digested tissues through a 70-µm cell strainer (Becton/Dickinson, Franklin Lakes, NJ). Single cell suspensions were seeded in 60 or 100 mm culture dishes and maintained in a humidified atmosphere with 5% CO 2 at 37 C. Colony forming units of cells were normally observed within 1 to 2 weeks after cell seeding and were passaged at 1:3 ratio when they reached ~80% confluence. Heterogeneous populations of these hdpscs were frozen and stored in liquid nitrogen at passages Stimulation and exposure to LPS Cells were thawed in a 37 C water bath and expanded for experimentation at passage 3. Cells were seeded onto 12-wells plates at a density of 5000 cells/ml and maintained in growth medium that consisted of α-minimum Essential Medium (α-mem; Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (FBS), 100 units/ml penicillin-g and 100 µg/ml streptomycin (Invitrogen) in a humidified atmosphere of 5% CO 2 at 37 C. At 90% confluence, this media was replaced with dentinogenic stimulation media, which consisted of original growth media supplemented with 2 mm KH 2 PO 4, 20 mm HEPES (Sigma, St. Louis, MO), 100nM dexamethasone prepared in α-mem, and 5 x 10-8 M 1,25-dihydroxyvitamin D 3 prepared in ethanol (all from Sigma). Cells were maintained in the dentinogenic media for 7 weeks, with a media change every 2 or 3 days. They were then exposed to 5, 10, or 20 µg/ml of P. gingivalis LPS (#05H23-SV) (InvivoGen, San Diego, CA) for 48 hours prior to the extraction of their 15

25 RNA. The negative control comprised of unstimulated cells maintained in growth media throughout the experiment without exposure to LPS. 2.3 Blockade of TLR2 pathway To study the effect of intra- and extracellular TLR2 blockade on DSPP and OCN gene expression prior to LPS exposure, cells stimulated with the dentinogenic media were incubated with either 25 mg/ml of anti-tlr2 neutralizing antibody, an LPS recognition blocker (Abcam, Cambridge, MA), or SC nM, prepared in 0.2% dimethyl sulfoxide (DMSO) (Cayman Chemical, Ann Arbor, MI) (Fig. 1A), a pharmacological inhibitor of IKK, a downstream component of the TLR2 recognition pathway (Fig 1B). These groups of cells were stored in a humidified atmosphere of 5% CO 2 at 37 C, for 1 hour, prior to the addition of 20 µg/ml P. gingivalis LPS to media in the culture well. Subsequently, they were maintained in a humidified atmosphere of 5% CO 2 at 37 C for 48 hours prior to the extraction of RNA to assess DSPP and OCN gene expression. Cells exposed to anti-tlr2, SC514 or 0.2% DMSO but not to LPS served as a positive control. 2.4 RNA extraction and cdna preparation Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA) and eluted to a final volume of 30 µl of sterile water, as recommended by the manufacturer. Cells were disrupted using 1% 2-β-mercaptoenthanol in RLT buffer with cell scrapers then vortexed for 3 x 10 seconds. Genomic DNA was eliminated using the RNase-Free DNase treatment (Qiagen). The concentration and purity of the samples were then verified with a Nanodrop 1000 Spectrophotometer (Thermo Scientific, Rockford, IL) and the samples stored at -80 C until used. Ten random samples were analyzed for RNA quality with a 2100 Bioanalyzer (Agilent Technologies, Inc, Santa Clara, CA) (see Figure 2). A 1 µg aliquot of the extracted RNA was used for oligo(dt) reverse transcription to obtain cdna with SuperScript II Reverse Transcriptase (Invitrogen). 2.5 Real-time quantitative PCR (RT-qPCR) 16

26 Human primer sets for DSPP, OCN and GAPDH, a control gene, were designed using Basic Local Alignment Search Tool (BLAST)-assisted internet search of a non-redundant nucleotide sequence database (National Library of Medicine, Bethesda, MD). The program MFOLD ( was used to analyze the amplicon for potential secondary structures that could prevent efficient amplification (see Figure 3). Primers were then synthesized (ACGT Corporation Toronto, ON) as shown in Table 1. Each 20 µl of reaction mixture contained 5.0 µl of cdna, 1 µl (10 µmol/l) of forward and reverse oligonucleotide primers, 10.0 µl of SsoFast EvaGreen Supermix (Bio-Rad, Mississauga, ON) and 3 µl of RNase-free water. Quantification of results from the real-time qpcr assays were analyzed with the CFX Manager (Bio-Rad). To ensure that primers annealed efficiently to their targets, a range of temperatures near and at the calculated melting temperature was tried, using a Bio-Rad thermal cycler with a temperature gradient feature. The optimal annealing temperature was determined to be 63 C. Care was taken to prevent nonspecific annealing and primer-dimer formation during the test procedure. A standard curve was used to determine reaction efficiency with a 2-fold dilution of cdna over seven points. A 1:16 cdna dilution yielded optimal PCR efficiency for all primer pairs. To check reaction specificity, the PCR product was analyzed by running samples on a 2% agarose gel, which displayed a single band (see Figure 4) and by examining the melt curve of the qpcr report, which displayed a single peak (see Figure 5). 2.6 Data analysis The experiments were performed in triplicate and the means and standard error calculated. Descriptive data and statistical analyses were performed utilizing the SPSS 16.0 software package (SPPS Inc. Chicago IL). Results were recorded as a ratio of normalized fold expression to GAPDH (2 Ct(gene of interest) /2 Ct (GAPDH) ). One-way analysis of variance (ANOVA) and post-hoc Tukey s test were performed to explore a statistical difference in gene expression among groups. All statistical analyses were two-tailed and interpreted at a 5% level of significance. 17

27 Chapter 3 Results 3.1 Characterization of isolated hdpscs hdpscs were isolated from normal human dental pulps typically formed colonies as shown in Fig 6A (43,132). After cell expansion, they were stimulated with dentinogenic medium for 7 weeks. Matrix production and mineralization of the cultures were observed by the presence of red stain (Fig 6B), indicating their potency in differentiating into dentingenic cell lineages upon stimulation. These hdpscs were used in our studies to determine the effects of P. gingivalis byproduct on their dentinogenic gene expression following differentiation (43,132). 3.2 Effect of P. gingivalis on DSPP and OCN gene expression To determine whether P. gingivalis by-products affect the activity of hdpscs differentiated into dentinogenic lineage, we first allowed cells to grow under the dentinogenic differentiation condition for 7 weeks. In pilot studies, dentinogenic gene markers DSPP and OCN were upregulated significantly after stimulation for such a time period. To observe the effect of P. gingivalis by-product on the expression of DSPP and OCN, the by-product was added to the cells at 7 weeks post-dentinogenic stimulation. Expression of DSPP (6.32 ± 2.47) and of OCN (4.70 ± 1.6) of the stimulated hdpscs was significantly higher (P < 0.05) than that of the unstimulated cells (Fig. 7). Exposure of stimulated cells to 5, 10, and 20 µg/ml by-product produced a dose-dependent decrease in DSPP and OCN gene expression (Fig. 7) When compared to the control gene (GAPDH), DSPP expression decreased to 3.19 ± 0.18, 2.60 ± 0.49, and 1.15 ± 0.29 respectively, and OCN expression decreased to 3.51 ± 1.18, 2.60 ± 0.67, and 1.66 ± 0.89 respectively. Only after exposure to 20 µg/ml of by-product, the expression of DSPP (1.15 ± 0.29) and of OCN (1.66 ± 0.89) was significantly lower than that of the unexposed stimulated cells (P < 0.05) and not significantly different from that of the unstimulated cells. 18

28 3.3 Effect of TLR2 blockade on suppression of DSPP and OCN gene expression DSPP and OCN gene expression of cells exposed to anti-tlr2, SC514, or 02% DMSO alone (results not shown) did not differ significantly from the levels recorded for the stimulated cells not exposed to by-product (Figs. 8, 9). Using the blockade of the TLR2 pathway by anti-tlr2 neutralizing antibodies (Fig. 8) and the pharmacological inhibitor SC514 (Fig. 9), gene expression of DSPP (4.67 ± 0.97, and 5.29 ± 1.66 respectively) and OCN (5.25 ± 1.69, and 5.82 ± 2.38 respectively) after exposure to 20 µg/ml by-product was significantly (P<0.05) higher than that of the stimulated, P.gingivalis-exposed cells without the blockade (1.16 ± 0.26). Both gene expression levels did not differ significantly from those of the stimulated cells not exposed to P. gingivalis by-product. 19

29 Chapter 4 Discussion In recent years, interest in regenerative endodontics as an approach to the treatment of infected immature teeth has been rekindled (35). This has been fueled by a failure of current methods of treatment to provide a favorable long-term outcome, and encouraging advances seen in stem cell research in related fields (10). Research is currently underway to develop a means by which the pulp space can be successfully re-populated with cells that, when stimulated, proliferate, differentiate, and re-initiate the dentinogenesis arrested when infection occurred (10,42,143). DPSCs appear to exist in inflamed pulp and retain their dentinogenic properties suggesting that inflamed pulp may have the potential to regenerate new pulp and re-initiate dentinogenesis upon successful disinfection of the pulp space (11). Therefore, one of the objectives of this study was to highlight the need to address infection in regenerative endodontics. Cell culture has become the principal tool in the study of cell responses to stimulating agents (144). In regenerative endodontics, hdpscs have been the dental stem cell source most studied (10). To date, no immortalized cell lines are available, so cells must be collected from the pulps of human teeth and kept in frozen storage until used. Unlike immortalized stem cell lines that retain their characteristics over multiple cycles, primary cell extracts remain vital but lose their potential to differentiate after a certain number of passages (43). For hdpscs, it was noted that this potential was lost after 4 passages (43). The hdpscs used in the present study were obtained from Dr. George Huang (in accordance with protocols approved by an ethics committee) from the pulps of healthy adult patients attending the University of Maryland Dental clinic. They have been used in several previously published studies (43,132,145). They are a mixed population of cells that include mesenchymal cells, fibroblasts and odontoblast progenitor cells. Different agents have been used as media supplements to stimulate hdpscs along an odontoblastic lineage. Dexamethasone was added to the list in 2005 after Alliot-Licht et al (146) showed that when added to media it could induce stem cells to express DSPP, a major odontoblastic cell marker. Previously dexamethasone had been added to stimulate expression of osteoblastic cell markers in human bone marrow stromal cells (147). Later vitamin D was also 20

30 added as a supplement (43) after it was shown to upregulate extracellular calcium deposition by hdpscs grown on dentin (43). Double stranded DNA is relatively stable compared to mrna, which varies in accordance with changes in cell environment (148). This makes RNA an excellent marker for assessing gene expression under changing conditions. Since mrna is sensitive to external stimuli, it is important that experimentation be conducted under controlled and well-defined conditions. Careful handling of the samples is essential to minimize variability among samples in each experimental group and provide the reproducibility necessary for scientific data. It also reduces the risk of contamination by ribonucleases (RNases). These enzymes break down RNA and are found in the environment including human skin and in dust particles (148). RNases are very stable, do not require co-factors, and are effective in small quantities (148). Although measures were taken throughout the study to ensure minimize exposure to RNases, some of the variability seen could still be expected and caused by the ubiquitous environmental present of the enzyme. RNA should be intact for reverse transcriptase PCR. In this study, RNA purity was measured using the Nanodrop, which assesses the sample with respect to protein and phenol contamination spectrophorometrically. An OD 260/280 ratio of 1.8 to 2.0 indicates good quality RNA that is devoid of protein and phenol contamination. All of the samples used in this study had OD 260/280 ratios between the specified values. In addition, deoxyribonucleases (DNases) were removed from the sample to ensure that their enzymatic activity would not influence RNA levels. RNA quality or integrity was assessed with Agilent s Bioanalyzer technology. The Bioanalyzer showed two distinct ribosomal peaks corresponding to 18S and 28S of eukaryotic RNA. Traditionally, the intensity of the bands on denaturing agarose gels have been used to calculate the ratio that served as an indication of RNA integrity. A 28S/18S ratio of two is considered to represent good quality (148). Denaturing gel electrophoresis requires a large amount of the RNA sample to detect a clearly visible band. The Bioanalyzer requires only a small amount (1.5 µl) of the RNA sample and therefore is a more practical way of determining RNA integrity. However, it does produce small variation in 28S/18S ratios. It was for this reason that Agilent incorporated a patented software, the RNA Integrity Number (RIN) that creates an algorithm based on the electrophoretic pattern and assigns a number from 1 to 10. A value of 10 represents the greatest integrity and 1, the poorest. In our study, values obtained were consistently about 9, which 21

31 ensured that the quality of RNA across samples was good. Because the RNA samples used in this study demonstrated good analyses from the Nanodrop and Bioanalyzer, there was confidence that a good sample was used for reverse transcriptase PCR. Both primer design and careful choice of target sequence are essential to ensure specific and efficient amplification of the gene. Target sequences ideal for RT-qPCR should be unique, base pairs long with a (guanine and cytosine) GC content between 50-60%, and should not contain secondary structures (148). It is recommended that primers should have a GC content of 50-60% and a melting temperature of C (148). Using the Primer Blast software, all primers and amplicons designed in this study met the above criteria. RT-qPCR was used to quantify gene expression patterns of DSPP and OCN with respect to a control gene, GAPDH. In this study, RT-qPCR a non-specific fluorescent dye that intercalated with double stranded DNA was used to assess relative gene expression. To ensure that the results obtained truly reflected the distribution of the target RNA, samples were prepared and stored in accordance with manufacturer s guidelines. Cells express levels of a specific mrna in response to their environment. In order to robustly detect and quantify the level of mrna present in a cell, the RNA must be amplified to acquire readable levels. RT-qPCR permits measurement of the amplification during PCR in real time (i.e. the amplified product is measured at each PCR cycle). The data thus generated can then be analysed by computer software to calculate relative degree of gene expression. To accurately quantify this gene expression, the RT-qPCR readings for the gene of interest is divided by the amplification recorded for the housekeeping or reference gene taken from the same sample. This normalized possible variations in the amount and quality of target RNA among samples. Throughout the study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping reference gene. GAPDH is a metabolic enzyme that is involved in the breakdown of glucose. Since the expression of GAPDH remained relatively constant across all samples, it allowed for an accurate comparison of target gene (DSPP and OCN) levels across samples. Variations in these target gene levels were therefore attributed to the influence of the stimulatory agents present in the media. This would have been negated if variations in the expression of GAPDH had been observed. 22

32 Normal pulp is a soft tissue of ectomesenchymal origin composed of stem cells, immune cells, ectomesenchymal cells, fibroblasts, preodontoblasts (149,150) and odontoblasts, supported by a vasculature, and a neural network in a gelatinous extracellular matrix. The odontoblast is a highly specialized end cell that forms the dentin. Other pulp cells appear to have the potential to ultimately differentiate into odontoblast- like cells and secrete hard tissue forming proteins, if properly stimulated. It has been estimated that approximately 1% of pulp cells retain this ability (151) and it is these cells that reportedly compose the cellular pool for de novo dentin formation. Collectively, cells with this dentinogenic potential have been referred to as odontoblast progenitor cells; and while the sites in the pulp from which these cells can be recruited have not been identified, it is known when stimulation, they express of a number of dentinogenesisassociated genes. Two of these genes are OCN, and DSPP ( ). The latter is reported to be a specific odontoblast cell marker (131). It was for this reason that expression of these genes by hdpscs used in the study were selected as a measure of dentinogenic potential and their suppression as a measure of LPS toxicity. Variances in the expression during the different phases of the study would therefore reflect the positive and negative effects of the procedures undertaken. One of the factors identified as an impediment to the successful clinical application of regenerative pulp procedures is the persistence of microorganisms and their by-products in the infected root canal after attempts have been made to clean and disinfect it. Currently used methods of endodontic disinfection are relatively more effective in eliminating microorganisms from the root canal than in eliminating their by-products, particularly LPS, a surface molecule of Gram-negative bacteria. LPS has been found in the root canal, in apical tissues, and in dentin subsequent to endodontic infection ( ). The molecule is potentially harmful to host cells as a toxin and as an immune-stimulant (24,158). P. gingivalis by-product was used in this study because this microorganism has been identified as a putative endodontic pathogen prevalent in endodontic infections (134) and because its immuno-stimulatory effects on hdpscs has yet to be investigated. Previous studies had shown that immune-stimulation (137,139), adversely affects cell function and survival in other cell lines (138). Reports have also been published to indicate that host recognition of P. gingivalis LPS was different than that of cells exposed to LPS derived from other Gram-negative microorganisms 23

33 found in the root canal microorganisms (106,159) whose LPS is mediated by TLR4. Indeed, the lipid A component of P. gingivalis is unique when compared with enteric lipid A (160,161). Much evidence has been presented indicating that P. gingivalis LPS and its lipid A are TLR2 ligands, not TLR4 ligands (162,163). However, since starting this study, novel reports have been published that demonstrate that irrespective of the structural heterogeneity of lipid A species in P. gingivalis LPS preparations, host recognition is mediated by TLR4 and not TLR2 (108). In fact, the TLR2-active component contained in the P. gingvialis LPS was separated and purified, and its chemical structure was shown to be a lipoprotein consisting of three fatty acid residues (108,164). That study was undertaken to elucidate the heterogenic differences of P. gingivalis LPS lipid A component and the contamination of the biologically active components of P. gingivalis. Ogawa et al (105) were the first group to determine the structure of P. gingivalis LPS lipid A in This group was also the first to chemically synthesize the counterparts of lipid A and verify that the biological properties indicate it to be solely a TLR4 agonist (108,164) suggesting that the results observed in this study are likely to do P. gingivalis lipoprotein contamination. Residual LPS after chemomechanical debridement has been well documented (24,133). Although residual lipoprotein has not been measured after root canal debridement, its has been reported in endodontic infections (165). Nonetheless, the effect of the P. gingivalis by-product used in this study did act on TLR2 pathway and affected gene expression of DSPP and OCN by hdpscs. If this receptor was not inactivated or its recognition blocked, in the presence of P. gingivalis by-product, it could adversely affect hdpscs. As was expected in this study, exposure of hdpscs to P. gingivalis by-product, induced a dose-dependent reduction in DSPP and OCN gene expression, reducing the potential of these cells to form dentin even when adequately stimulated. The pattern was similar to one seen in a previous study that assessed DSPP and Runx2 gene expression in a rat dental pulp cell line exposed to A. actinomycetemcomitans LPS (128). In this study, we confirmed that exposure of hdpscs to the P. gingivalis by-product led to decreased gene expression of DSPP and OCN. We have also demonstrated that an extracellular and intracellular blockage of the P. gingivalis by-product recognition receptor of TLR2 could inhibit this effect. This was the first time that such activity had been recorded in a hdpsc population. Further studies are necessary to strengthen the results. 24

34 The mixed cell population that was used is known to contain odontoblastic precursor cells. After dentinogenic stimulation an increase in gene expression of DSPP, an odontoblast-specific gene (131) was upregulated as was OCN, a protein found throughout human odontoblasts (131). Of the cells that exist in the pulp, odontoblasts and odontoblast precursor cells are the only ones that express DSPP and OCN; thus dentinogenic stimulation affected these cells. Because the cell population was mixed, it is impossible to determine what affect the stimulation had on other cells in the population. The evidence could be strengthened if a pure cell population was used in this study. The results reported in this study are not intended to oversimplify the major obstacle posed by microorganisms and their by-products in the development of effective regenerative endodontic techniques. The microbiota of the infected root canal is highly diverse (166,167) as are the types and concentrations of LPS that can persist after root canal debridement and disinfection. In vitro studies such as this only address a single and detached issue of a very complex clinical problem and do not delve into the many interactions that can occur during clinical infections. A study involving multiple bacterial species infections would prove to be more characteristic of an in vivo situation. Interspecies interaction may alter the virulence of certain strains bacteria and in combination a synergistic effect or hindering effect may be seen. Since LPS derived from most Gram-negative root canal microorganisms activates TLR4 receptors (106,112,168), a blockade of both classes of receptors appears to be necessary if all inflammatory mediators that induced pro-inflammatory cytokine production is to be moderated. In addition to further investigate the host recognition of P. gingivalis, using a neutralizing TLR4 antibody to compare to the results of this study should also be undertaken. Alternatively, pretreating cells with SC514, the common pharmacological inhibitor of the IKK (proinflammatory pathway for TLR2 and TLR4) prior to adding a variety of LPS and lipoprotein to hdpscs could also provide insight as to whether the blocking agent has potential as a therapeutic agent in regenerative endodontics. In addition, to investigate the blocking agents used in this study, using lipopeptide from putative endodontic pathogens that activates TLR2, could provide further insight into the interaction between TLR2 activation and expression of genes involved in mineralized matrix proteins. 25

35 Chapter 5 Future Directions Future direction to develop an immortalized cell line to be used in studies such as this one, or studies that investigate the characterization of hdpscs would be helpful. However, until such a cell line is established, another approach to strengthen the results obtained in this study is to study the gene expression of other components involved in mineralized matrix formation. An assessment of protein expression rather than gene expression would appear appropriate to ensure that DSPP and OCN are actually synthesized, since the presence of mrna does not necessarily mean that the proteins are present. However, protein quantification to demonstrate DSPP and OCN presence may prove difficult as their translated parts become secreted into the extracellular matrix and are therefore difficult to measure. In addition, gene assays tend to be more sensitive than protein assays, in that very small amounts of transcript are required to detect the presence. Proteins on the other hand generally require much larger volumes. Culturing the cells on glass and applying a fixative to stain them prior to applying a specific antibody for OCN protein or products of DSPP translation and post-modification (e.g. DGP, DSP, or DPP) could be one way to analyze the protein expression. Another future approach should also incorporate a study of the system in a three-dimensional in vitro model. Although cell culture is a valuable tool the initial investigations of hdpscs behaviour, the characteristics on a two-dimensional environment may enhance the cells ability to obtain nutrients from the environment. 26

36 Figure and Table Legend Figure 1. A. Molecular structure of pharmacological inhibitor SC514 (Cayman Chemical, Ann Arbor MI). MF: C 9 H 8 N 2 OS 2. SC514 is a selective and reversible inhibitor of IκB kinase (IKK); B. One of the reported intracellular pathways downstream of Toll-like Receptor 2 activation (Adapted from Strober et al (169)). Figure 2. Bioanalyzer electropherogram of total RNA. Total RNA run on Agilent s 2100 Bioanalyzer shows two distinct ribosomal peaks corresponding to 18S and 28S for eukaryotic RNA. A perfect quality RNA sample would normally yield a 28S/18S ratio of 2 and is a reasonable way to estimate RNA integrity. However it is not ideal. Agilent has introduced a software algorithm that takes the entire electrophoretic trace into account, termed the RNA Integrity number, which is based on a numbering system from 1 to is the most degraded and 10 is the most intact. This interpretation ensures better reproducibility. A. Unstimulated hdpscs B. Stimulated hdpscs C. Stimulated hdpscs exposed to LPS Figure 3. MFOLD analysis of the amplicons for potential secondary structures. The program MFOLD ( was used to analyze the amplicon for potential secondary structures that could prevent efficient amplification. The images represent the folding that would take place at 63 C, which demonstrate low secondary structure formation. A. hgapdh 97 bps B. hdspp 277 bps C. hocn 155 bps 27

37 Figure 4. Gel analysis of PCR products. Real-time quantitative PCR products run on a 2% agarose gel. Single band at the corresponding size confirms the specificity of the primers. Lanes 1-7 represent the Fermentas 100 bp ladder, the RT-qPCR product from unstimulated cells, stimulated cells, stimulated cells exposed to LPS 20 µg/ml, stimulated cells pretreated with SC nm, stimulated cells pretreated with SC nm and exposed to LPS 20 µg/ml, stimulated cells exposed to 2% DMSO respectively. Figure 5. Melt curve for real-time quantitative PCR results. A single peak on the melt curve analysis indicates a single PCR product. A. hgapdh has a single peak at 82.5 C B. hocn has single peak at 87.5 C Figure 6. hdpscs grown and analyzed in cultures. A. Typical clonogenic hdpscs forming a colony in the culture dish after being isolated from human pulp tissues using enzyme digestion. B. A representative image showing cultured hdpscs underwent dentinogenic differentiation after being stimulated by media containing dexamethasone and 1,25-dihydroxyvitamin D 3 for 7-8 weeks. Mineralized particles were stained red by Alizarin Red S. Figure 7 Effect of P. gingivalis on DSPP and OCN gene expressions. Stimulated cells were exposed to P. gingivalis derived by-product at final concentrations of 5, 10, or 20 µg/ml for 48 hrs. Quantitative real-time PCR was used to detect mrna expression levels of DSPP (A) and OCN (B) normalized to GAPDH. Experiment was performed in triplicate and error bars represent standard error of the mean. Statistical significance denoted by similar letter designation (P < 0.05) using ANOVA and post hoc Tukey s tests. Figure 8 28

38 Effect of anti-tlr2 on P. gingivalis suppression of DSPP and OCN gene expression. Stimulated cells were incubated with anti-tlr2 prior to exposure to 20 µg/ml of P. gingivalis by-product for 48 hours. Quantitative real-time PCR was used to detect mrna expression levels of DSPP (A) and OCN (B) normalized to GAPDH. Experiment was performed in triplicate and error bars represent standard error of the mean. Statistical significance denoted by similar letter designation (P < 0.05) using ANOVA and post hoc Tukey s tests. Figure 9 Effect of pharmacologic inhibitor SC514 on P. gingivalis suppression of DSPP and OCN gene expression. Stimulated cells were incubated with SC514 prior to exposure to 20 µg/ml of P. gingivalis by-product for 48 hours. Quantitative real-time PCR was used to detect mrna expression levels of DSPP (A) and OCN (B) normalized to GAPDH. Experiment was performed in triplicate and error bars represent standard error of the mean. Statistical significance denoted by similar letter designation (P < 0.05) using ANOVA and post hoc Tukey s tests. Table 1 Primer sequences for the genes analysed. 29

39 Figures and Table Figure 1 A. B. 30

40 Figure 2 A. B. 31

41 C. 32

42 Figure 3 A. hgapdh 97 bps B. hdspp 277 bps C. hocn 155 bps 33

43 Figure 4 A. hgapdh 97 bps B. hdspp 277 bps C. hocn 155 bps 34

44 Figure 5 A. B. 35

45 Figure 6 A. B. 36

46 Figure 7 A. B. 37

47 Figure 8 A. B. 38

48 Figure 9 A. B. 39