UAB Alumni UAB Alumni UAB Alumni Nair 2004

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1 Bacteria in Dentinal Tubules Extraradicular Infection Consensus: infection is primarily inside canal! -!good evidence from SEM and TEM studies! -!culturing is prone to false positives Upside: treatment rationale! -! intraradicular infection is much more accessible! -!non-surgical retreatment has high success When is extraradicular infection present! -! acute abscess, chronic abscess! -! special bacteria: Actinomyces, Propionibacterium Anatomy: where does the canal end?! -!bacteria may lodge in apical canal parts, close to!! the end of the root canal Sundqvist und klinisches Bild mit guter WF Nair 2004

2 How Do Bacteria Attack? With virulence!! -!They come in VERY big numbers! -! They have several unpleasant abilities How Do Bacteria Attack? With strategy!! -!They arrange in biofilms How Do Bacteria Attack? With strategy!! -!They arrange in biofilms (movie from Singh et al, Nature 417, 2002)

3 The Silver Bullet: antibiotics? Pain relief Antibiotic Myths Bacterial spectrum Non-vital pulps

4 Antibiotic Facts Pain relief! - antibiotics are no painkillers! - ibuprofen is the most effective pain medication Bacterial spectrum! - broad-spectrum ABs should be avoided! -!oral penicillin has a good spectrum Non-vital pulps! - treatment of infected root canals should normally! be accomplished without ABs Regenerative Endodontics What is it?! - return of a vital response after nonvital response - in this context: reconstitution of functional pulp What are the goals and benefits! - deposition of hard tissue for immature teeth - immunologically competent functional tissue What are the indications! - currently: teeth with large apical foramina - research is underway to extend the spectrum

5 Regenerative Endodontics Stimulation vs. delivery! - removal of cells and tissues, recruitment of undifferentiated cell populations Metzger 2007 Regenerative Endodontics Stimulation vs. delivery! - autologous cells, apical papilla cells - stem cells from other sources Scaffold! - collagen, gel etc. - fibrin matrix Crucial: Blood supply! - VEGF and other key factors - transportation of bacteria/debris into periapical area Regenerative Endodontics Preop 9m recall Case by Dr. Patrick King

6 Case 1 10 year old female presented to the pedo clinic with pain to biting on the upper right side Radiograph reveals decay approximating the pulp Cold (-), Percussion (+), Mobility (1), throbbing pain PARL possibly developmental or pathosis Case by Dr. Nick Morton Upon access necrotic pulp Irrigation w/17% EDTA triple antibiotic paste (minocycline/cephlexin*/ metronidazole) placed to WL Minocylcine is now substituted with cipro (staining) Cotton and GI temporary Case 1 Patient returned after 1 month. No longer symptomatic, tooth was no longer mobile Following re-access bleeding was induced and allowed to clot. (wait 15 minutes) MTA placed against the clot and tooth permanently restored with dual cure composite Case 1

7 Final restoration Final radiograph 1 st appointment Final Rx Case by Dr. Nick Morton Injuries to Permanent Dentition Symposium Considerations for Regeneration Procedures Alan S. Law, DDS, PhD Abstract When pulp tissue becomes necrotic in immature teeth, the prognosis of the teeth is compromised. Disinfection of the root(s) presents several challenges including difficulties in cleaning and shaping large canals with open apices, obturation of canals with open apices, and potential root fractures caused by thin and/or weakened root walls. Regenerative endodontic procedures may increase the prognosis of the compromised immature tooth by re-establishment of a functional pulp tissue that fosters continued root development and immune competency. This article reviews the literature related toanddiscussconsiderationsforregenerativeendodontic procedures and how these procedures may increase the prognosis for immature teeth with necrotic pulp tissue. (J Endod 2013;39:S44 S56) Key Words O Chlorhexidine, considerations, discoloration, immature, mineral trioxide aggregate, necrotic, outcomes, radiograph, regenerative endodontics, revascularization, revitalization, sodium hypochlorite, tooth, triple antibiotic paste From The Dental Specialists, Lake Elmo, Minnesota; and the Division of Endodontics and Graduate Endodontics, University of Minnesota, School of Dentistry, Minneapolis, Minnesota. This article is being published concurrently in Pediatric Dentistry 2013;35(2). The articles are identical. Either citation can be used when citing this article. Address requests for reprints to Dr Alan S. Law, The Dental Specialists, 8650 Hudson Boulevard, Lake Elmo, MN address: endolaw@comcast.net /$ - see front matter Copyright ª 2013 American Academy of Pediatric Dentistry and American Association of Endodontists. ne of the goals of endodontics is keeping the dentition in a physiologically functional state for the maintenance of oral and systemic health. Trauma to the developing dentition, when it results in pulp necrosis and incomplete root formation, can lead to a premature loss of permanent teeth, which may compromise the dentition. Teeth with immature apices present challenges in cleaning and shaping large canals with blunderbuss apices, obturationof canals with open apices, and potential root fractures caused by thin and/or weakened root walls (1 3). By regenerating tissue in the canal space of immature teeth, regenerative techniques can help to restore the physiologically functional dentition. The concept of revitalizing tissue in the canal space has been around for several decades.acase series by Nygaard-Ostby and Hjortdal in 1971 (4) showed ingrowth of fibrous connective tissue and cementum in mature teeth with previously necrotic pulp tissue. In 1974, Myers and Fountain (5) reported an increased root length and calcified material in necrotic canals of monkey canines with immature apices after disinfection with NaOCl and filling the canals with citrated whole blood or gel foam. In 1976, Nevins et al (6) showed that the disinfection of necrotic canal space in immature monkey incisors followed by the placement of a gel (containing collagen, calcium chloride, and dipotassium hydrogen phosphate) and short obturation with gutta-percha could result in various forms of hard and soft connective tissue including cementum, bone, and reparative dentin lining the walls of the root canal. Although the findings of the 3 reports showed the possibility of creating new tissue in necrotic canal space, it was not until the past decade that there has been an increased interest in applying these concepts in clinical endodontics. Two case reports can be credited with sparking recent interest in regenerative endodontics. The first, which was by Iwaya et al (7), showed that NaOCl and H 2O2disin- fection of necrotic tissue in an immature premolar followed by canal medication with metronidazole and ciprofloxacin resulted in continued root formation and clinical evidence of renervation. The second, by Banchs and Trope (8), showed a similar outcome of continued root development and renervation after canal disinfection with NaOCl and chlorhexidine and the placement of triple antibiotic paste (TAP; ie, ciprofloxacin, metronidazole, and minocycline). Both of these case reports showed 3 important principles of regenerative endodontic procedures: 1. Elimination of bacteria from the canal system 2. Creation of a scaffold for the ingrowth of new tissue 3. Prevention of reinfection by creating a bacteria-tight seal Although these 2 case reports showed clinical evidence of continued root growth, they did not show histology of the canal contents.as a result, it is not clear what type of tissue was present. There have been several terms used to describe the introduction of newlivingtissue intothecanal space. Theseinclude regeneration, revascularization, and revitalization. There has been debate as to which of these 3 terms (ie, revascularization, revitalization, regeneration) is most appropriate to the outcome of procedures usedtoregeneratepulptissue (3, 9, 10). Thetermrevascularizationdescribes the re-establishment of the vascular supply to existing pulp in immature permanent teeth (11). Revitalization describes the ingrowth of tissue that may not resemble the original lost tissue (12). Endodontic regeneration is the replacement of damaged structures, including dentin and root structures, as well as cells of the pulp-dentin complex (13). More recently, 2 case reports (14, 15) have included histologic sections of teeth that were extracted after regenerative endodontic treatment. The findings from these reports may give a glimpse of what may be occurring in other teeth. Torabinejad and Faras (14) presented clinical, radiographic, and histologic findings S44 Law JOE Volume 39, Number 3S, March 2013 Clinical steps! -! indication, prognosis! -!anesthesia - 1 st appointment irrigation disinfectant paste scaffold - 2 nd appointment pulp space barrier definitive filling Law 2013 Colleagues for Excellence Spring 2013 Regenerative Endodontics Published for the Dental Professional Community by the American Association of Endodontists. This issue sponsored by a grant from the AAE Foundation. Clinical steps! -! indication, prognosis! -!anesthesia - 1 st appointment irrigation disinfectant paste scaffold - 2 nd appointment pulp space barrier definitive filling Cover artwork: Rusty Jones, MediVisuals, Inc.

8 Indication Considerations - large apical diameter, young patients Diagnosis - pulp necrosis, sinus tracts Expected outcomes - favorable / unfavorable Clinical steps - disinfection, scaffold, definitive closure Apical barrier Expected Outcomes - radiographically visible, apical stop for fill Root Length Wall Thickness Bose 2009

9 Regenerative Endodontics Nature of the deposited hard tissue - strengthening effect not clear - hard and soft tissues are important Paqué, 2011 Predictability - treatment outcome may depend strongly on conditions - no clear clinical recommendation for procedure Conclusion - vital pulp therapy may become attractive? - significant development potential J Endod 2011, 37: Histologic Characterization of Engineered Tissues in the Canal Space of Closed-apex Teeth with Apical Periodontitis Jo~ao Eduardo Gomes-Filho, PhD,* Paulo Carvalho Tobias Duarte, PhD,* Edilson Ervolino, PhD, Suely Regina Mogami Bomfim, PhD, Caio Jose Xavier Abimussi, MSc, Ludmilla Mota da Silva Santos, MSc,* Carolina Simonetti Lodi, PhD,* Sandra Helena Penha De Oliveira, PhD, Eloi Dezan, Jr, PhD,* and Luciano Tavares Angelo Cintra, PhD* Abstract Introduction: The aim of this study was to investigate the capacity of endodontic regenerative procedures combining an induced blood clot, platelet-rich plasma (PRP), and bone marrow aspirate (BMA) to regenerate dental pulp in canine closed-apex necrotic teeth. Methods: Apical periodontitis was induced in 20 upper and lower premolars of 2 dogs. After biomechanical preparation, enlargement to a #60 file, and disinfection with a triantibiotic paste for 28 days, the roots were randomly assigned to 4 treatment groups: blood clot (BC), BC + PRP gel, BC + BMA gel, and BC + BMA/ PRP gel. Negative controls were also included. After a 3-month follow-up period, the animals were killed. Results: Histologic analysis showed the presence of newly formed vital tissues (connective, cement-like, and bone-like tissue) in 23 of the 32 treated roots (71.87%). There was no statistically significant difference between the treatment groups. Conclusions: New vital tissues were formed and characterized as connective, cementum-like, or bone-like, but not as pulp-like tissue; PRP and/or BMA did not improve the tissue ingrowth. (J Endod 2013;-:1 8) Key Words C Bone marrow aspirate, necrotic teeth, platelet-rich plasma, regenerative endodontics, tissue engineering, vital tissue growth Basic Research Technology omplete restoration of a functional dentin-pulp complex cannot be achieved by conventional endodontic treatment (1, 2). However, with the advent of regenerative medicine, it became possible to regenerate pulp through alternative endodontic regenerative procedures (ERPs) (3). These procedures are based on the principles of tissue engineering that aim to develop new tissues to replace lost or malfunctioning organs by using a source of stem cells, a three-dimensional scaffold for the growth of these cells, and signaling molecules (1, 3). The most common ERP uses blood clots and cell transplantation. In a number of studies, the induction of a clot alone did not promote pulp regeneration in immature necrotic dog teeth (4 6), prompting the investigation of new ERPs that modify the clot or do not use it at all. Platelet-rich plasma (PRP) has been investigated in the context of human ERPs (3). A patient treated with PRP showed the presence of pulp tissue and a lack of bone tissue (7). However, no pulp regeneration was achieved by injecting PRP, dental pulp stemcells (DPSCs), or a mixture of both into the roots of mature dog teeth without inducing a clot (8). Moreover, in a human subject, injection of PRP and blood clot caused an increase in root length and thickness compared with that observed with the induction of a blood clot alone (9). Dog pulp regeneration has been achieved by using stem cell transplantation (10 12). However, cultivation and expansion have been linked to reduced viability, selection, and unwanted reprogramming and/or cellular dedifferentiation (13). In addition, it is expensive, time-consuming, and associated with an increased risk of infection (14, 15). Moreover, as the pulp diminishes with age, alternative sources need to be evaluated (12). Bone marrow mesenchymal stem cells (MSCs) have been used as an alternative source for dog pulp regeneration (12). The use of bone marrow aspirate (BMA) can be a straightforward, low-cost, and fast method with a low risk of contamination (16, 17). An ERP for mature necrotic permanent teeth has been clinically investigated, and the resolution of apical radiolucency and regression of clinical signs and symptoms were observed at recall appointments (18). Moreover, histologic evaluation of transplantation of DPSCs and/or PRP into root canals showed no evidence of pulp tissue or even improved tissue ingrowth in mature vital dog teeth (8). However, no reported study has aimed to histologically investigate pulp regeneration of closed-apex necrotic teeth by using alternative ERPs. The aim of this study was to investigate the synergistic From the *Departments of Endodontics and Basic Sciences, Araçatuba School of Dentistry, Universidade Estadual Paulista, Araçatuba, S~ao Paulo; and Clinical, Surgery and Animal Reproduction, Araçatuba School of Veterinary, Universidade Estadual Paulista, Araçatuba, S~ao Paulo, Brazil. Supported by CNPq (National Research Council). Address requests for reprints to Dr Jo~ao Eduardo Gomes-Filho, Endodontics, Araçatuba School of Dentistry, Universidade Estadual Paulista, R. Jose Bonifacio, 1193 Araçatuba, S~ao Paulo, Brazil. address: jegomesfilho@yahoo.com.br /$ - see front matter Copyright ª American Association of Endodontists. JOE Volume -, Number -, Engineered Tissues in Teeth with Apical Periodontitis 1 Human histology: The tissues formed in the canal of revitalized human tooth are similar to cementum- or bone-like tissue and fibrous connective tissue. Shimizu 2013

10 Law 2013 First Appointment Case selection - post space needed, allergies, compliance? Consent - mostly minors - discoloration of the crown possible: TAP, MTA Access Disinfection - no instrumentation - irrigation with 1.5% NaOCl - place Ca(OH)2 or TAP/DAP Temporary filling Issues TAP/DAP - 1:1:1 minocyline, ciprofloxacin, metronidazole - staining due to minocycline, remove or seal dentin Ca(OH)2 - recent data shows less cytotoxic compared to TAP - readily available, place with lentulo spiral Temporary filling - should avoid recontamination - e.g., 4mm layer Cavit plus IRM

11 Basic Research Technology Direct Effect of Intracanal Medicaments on Survival of Stem Cells of the Apical Papilla Nikita B. Ruparel, DDS, MS, PhD,* Fabricio B. Teixeira, DDS, MS, PhD,* Caio C.R. Ferraz, DDS, MS, PhD, and Anibal Diogenes, DDS, MS, PhD* Abstract Introduction: Regenerative endodontic procedures are an alternative treatment for immature teeth with necrotic pulps. Typically, intracanal medicaments such as triple antibiotic paste (TAP) or double antibiotic paste (DAP) and calcium hydroxide (Ca[OH] 2) are used for disinfection. However, their effect on human stem cells of the apical papilla (SCAPs) is unknown. We hypothesized that intracanal medicaments at high concentrations are toxic to SCAPs. To test this hypothesis, a cell culture assay was used. Methods: Briefly, SCAPs were cultured and subjected to either no drug treatment or various concentrations including TAP, DAP, modified TAP (ciprofloxacin, metronidazole and cefaclor), Augmentin (Champs Pharmacy, San Antonio, TX), or Ca(OH) 2. Viable stem cells counts were obtained using an automated method of detecting trypan blue dye at 3 days after treatment. Results: All 4 antibiotics significantly reduced SCAP survival in a concentrationdependent fashion. Interestingly, Ca(OH)2 was conducive with SCAP survival at all concentrations. Conclusions: Collectively, our data show that high concentrations of antibiotics have a detrimental effect on SCAP survival, whereas lower concentrations as well as Ca(OH)2 at all tested concentrations are conducive with SCAP survival and proliferation. These studies highlight the clinically important point that intracanal medicaments must be used at concentrations that are bactericidal while having minimal effects on stem cell viability. (J Endod 2012;38: ) Key Words R Apical papilla, biocompatible, calcium hydroxide, dental stem cells, double antibiotic, human stem cells of the apical papilla, regenerative, triple antibiotic From the *Department of Endodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas; and Dentistry, State University of Campinas, S~ao Paulo, Brazil. Address requests for reprints to Dr Anibal Diogenes, Department of Endodontics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX address: Diogenes@uthscsa.edu /$ - see front matter Copyright ª American Association of Endodontists. Department of Restorative Dentistry, Piracicaba School of egenerative endodontic therapy has become a viable alternative in the treatment of immature teeth with pulpal necrosis. Since its inception, several successful case reports have been published (1 9) with substantial advancements in the understanding of the biological principles involved in the regeneration of the pulpdentin complex, including the evaluation of stem cells, growth factors, and signaling molecules involved in the differentiation of stem cells into various cell types (10 12). Moreover, it has become evident from preclinical studies and clinical case reports that a successful regenerative procedure of a tooth with necrotic pulp requires adequate disinfection of the root canal system. Clinical regenerative endodontic procedures depend primarily on chemical debridement of the canal space with minimal to no mechanical instrumentation (1 8). The most widely used intracanal medicament used in regenerativeendodontic/ revascularization procedures is the mixture of ciprofloxacin, metronidazole, and minocycline, which is also called triple antibiotic paste (1, 13, 14). This combination has been shown to be highly efficacious against bacteria commonly found in infected root canal systems in both in vitro (13, 14) and in vivo studies (1, 3, 5, 9, 15). However, other intracanal medicaments have been used including Ca(OH) 2 (4); formocresol (7); the combination of metronidazole and ciprofloxacin (double antibiotic paste) (2); and the combination of metronidazole, ciprofloxacin, and cefaclor (modified triple antibiotic paste) (8). Although the combination of amoxicillin and clavulonic acid (Augmentin; Champs Pharmacy, San Antonio, TX) has not yet been used in a published regenerative endodontic case, it has been found to be effective against 100% of endodontic bacteria (16), and it may represent an important medicament alternative for patients without a history of a penicillin-like drug allergy. Collectively, the antibacterial effects of these drugs are well known; however, there is a large gap in knowledge regarding the potential toxic effect on human mesenchymal stem cells. Thus, the aim of this study was to evaluate the effect of commonly used intracanal medicaments on the survival of human stem cells of the apical papilla (SCAPs) in vitro. Patient Recruitment Materials and Methods This study was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio. After verbal and written communication for informed consent, stem cells of the apical papilla were harvested from 2 extracted immature mandibular third molars (#17 and #32) from a 17-year-old female patient recruited from the clinic of the University of Texas Health Science Center at San Antonio School of Dentistry. The extracted teeth were immediately rinsed in sterile phosphatebuffered saline (PBS) and stored in a container with sterile PBS until SCAP harvesting. Harvesting of SCAP and Cell Culture SCAPs were obtained from the harvested apical papillae by enzymatic digestion as described previously (17). Cells in suspension were plated in 10-cm diameter poly-d lysine-treated culture plates (BD Biosciences, Bedford, MA). The cells formed single colonies and exhibited typical fibroblast-like morphology. Cells were allowed to expand in culture to 70% to 80% confluency followed by treatment with 0.05% trypsin (Gibco, Carlsbad, CA) and passing the culture to subsequent culture plates.scaps between the fifth and eighth passages were used in this study Ruparel et al. JOE Volume 38, Number 10, October 2012 Toxicity! -!TAP in the currently used way has high concentration - this is toxic to stem cell, impacting proliferation and differentiation - lower concentrations and Ca(OH)2 are preferred Timing Second Appointment - after 3-4 weeks, longer not advisable Anesthesia - no vasoconstrictor Reaccess and irrigation - EDTA rather than antimicrobials Scaffold - overinstrument to create bleeding, alt. collagen plug Tissue barrier - collagen app. 3mm below CEJ, MTA fill, alt. GIC Material & Methods! -! SCAP were isolated from third molars and characterized - human root segments were subjected to irrigation with either 17% EDTA; 6% NaOCl/EDTA; EDTA/2%CHX or NaOCl/EDTA/NaOCl/alc/CHX - root segments were filled with SCAP and PRP, cultured for 21 days and then processed for immunohistochemistry - cell viability was determined J Endod 2011, 37:

12 Effect of Irrigation on SCAP A B C D J Endod 2011, 37: Follow-up Clinical - no pain or tissue swelling - between the two primary appointment and at 6m recall Radiographic - apical lesion resolves (6-12m) - root wall thickness increase (12-24m) - root lengthening, apical closure (variable) Discoloration - sometimes esthetically compromising What to Expect: Public health impact - patient population currently small - extension to mature apices possible? Verma 2012 Research impact - expect to broaden biological knowledge base - will establish molecular methods in the clinic Strategies - vital pulp therapy may become attractive and feasible - significant development potential

13 MTA pulp caps! -!success rate varies from >90% to about 50% Vital Pulp Therapy Predictable? - for adults depends on pulpal status - tests insufficient: cold, electric Immature root development - reasonable alternative to RCT - MTA material of choice Strategies - avoiding pulp exposure preferrable - few well-done clinical studies

14 Research in RegEndo Clinical treatment is effective - for a select patient pool - potential to extend the spectrum Stimulate research environment - towards understanding endodontic biology - validation of current treatment modalities? Unintended consequences - developments of new pulp testing methods - disruptive strategies? Conclusions Long-range: two pillars - vital pulp therapy - minimal invasive conventional endodontics Transition period - gradual R & D for both - special cases: define indications and techniques Cognitive framework - establish best practices, currently insufficient evidence - socioeconomics and access to care Conclusions Elimination of microbes is key to success! -! presence of organisms linked to disease symptoms! -! both counts and virulence of colonies are important Efficacy of antimicrobial regime can vary! -! understanding chemical and biological interactions! -! assessing cases and their individual challenges Clinical strategies must vary! -! wide range of possibilities! -! technical and biological details

15 A Break? False Summit, Mt. Tam