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    Regenerative Endodontics - Cohen

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    Regenerative Endodontics ANIBAL DIOGENES CHAPTER OUTLINE STEPHANE SIMON | ALAN S. LAW Overview of Regenerative Dentistry Overview of Regenerative Endodontics Preclinical Studies on Regenerative Endodontics Stem Celis Growth Factors/Morphogens Scaffolds Delivery System Translational Studies Summary of Basic Research on Regenerative Endodontics OVERVIEW OF REGENERATIVE DENTISTRY ‘Advancements in tissue engineering are dramatically changing. medicine and dentistry. Tissue engineering is an imterdisciphin- ary field that applies the principles of engineering and the life sciences to restoring, maintaining, or replacing biologic func~ tion." It inwolves the interplay among stem cells, growth. factors, and scaffolds (biologic matrices). It has become ‘mereasingly clear that the intentional manipulation of these three factors can lead to the regeneration of tissue function that would not otherwise occur if repair had taken place without intervention." This relatively young field was frst applied to medicine with many examples of regenerative medicine approaches used in the clinical practice, """"*" Although the inclusion of tissue engineering in dentistry is more recent, it ts also fundamentally changing the way clinicians are treating patients while providing a ferule research field that fosters future advancements and therapies Most of the history of dentistry is marked by the evolution of dental materials and techniques tailored to the replacement of lost or diseased tissues with inert materials, This prosthetic replacement of missing dental tissues has prevailed in dentistry since the primordial examples of dental treatments in ancient civilizations." 7" In contrast, the goal of regenerative den- Lusty is to induce biologie replacement of dental ussues and their supporting structures, The potential for regenerative den- tistry is in large part due to advancements in biologic therapies that apply principles of issue engineering with the spatial and. temporal assembly of stem cells, growth factors, and scaffolds to achieve the functional regeneration of a missing tissue Pioneering work supporting the concept of regenerating dental tissues was reported in the 1960s when Dr. BW, Hermann described the application of calcium hydroxide (CalOH],) for vital pulp therapy"? and Professor Nygaard- sthy evaluated a revascularization method for reestablishing, Clinical Studies on Regenerative Endodontics Clinical Procedures Related to Regenerative Endodontics Overview of Clinical Regenerative Endodontic Procedures (REPS) Example of a Revascularization Protocol Clinical Measures of Treatment Outcome ‘Summary a pulp-dentin complex in permanent teeth with pulpal necrosis (discussed later)" The scope and clinical application of regenerative dental procedures have advanced to now include guided tissue or bone regeneration (GTR, GBR) procedures and distraction osteogenesis," the application of platelet rich plasma for bone augmentation,” Emdogain for regenera~ tion of periodantal tssues and pulp,” recombinant human bone morphogenic protein (ChEMP) for augmentation of boone," and clinical tials on the use of fibroblast growth factor 2 (FGF-2) for periodontal tissue regeneration *” The potential of regenerative procedures in endodontics has heen emphasized by elegant studies demonstrating the regeneration of pulp, dentin, and enamel using scaffold materials and stem *°Thus, regenerative dental procedures are emerg- ing asa vital, evolving field of dental care, creating a paradigm shiftin many dental specialties, including endodontics.”» This chapter reviews the cUrrent status of regenerative endodontic procedures with an emphasis on biologic principles and the advantages and limitations of currently available clinical procedures Overview of Regenerative Endodontics “The developing dentition is at risk for pulpal necrosts due to trauma, caries, and developmental dental anomalies such as dens evaginatus.”°' "8" Loss of an immature perma nent tooth in young patients with mixed dentition can be devastating, leading to loss of function, malocclusion, and inadequate maxillofacial development, These teth were tradi- tionally weated with apexifcation procedures using either Jong-ierm calcium hydroxide ceatment”” or immediate placement of a mineral trioxide aggregate (MTA) apical plug Although these treatments often resolve the signs and symp- toms of pathosis, they provide litle co no benefit fr continued root development.” Thus, immature teeth treated with these procedures are considered in a state of arrested development, 447 448 PART II The Advanced Science of Endodontics and no further root growth, normal pulpal nociception, and immune defense should be expected Regenerative endodontic procedures (REPS) have been defined as biologically based procedures designed to replace damaged structures such as dentin, root structures, and cell of the pulp-dentin complex." This new treatment modality has emerged as an alternative tha, im addition to healing apical periodontitis, aims to promote normal pulpal physiologic fune- tions. These include continued root development, immune competency, and normal nociception, as seen in some pub- lished cases." Thus, the ultimate goal of these procedures i to regenerate the components and normal function ofthe pulp: dentin complex. Regenerative endodontics is founded on the seminal work ‘of Dr. Nygaard-Ostby, completed sn the 1960s. He bypothe- sized that a blood clot could be the first step in the healing of a damaged dental pulp, similar tothe role af the blood clot in the healing process observe in other areas (eg, alveolar bone following extraction)" To test the bypothests that the pres fence of a blood clot within a root canal system promotes healing, mature teth diagnosed with either vital or necrotic pp received pulp space debridement followed by foraminal elargement, medicament dressing for the necrotic eases, ‘evoked sntracanal bleeding, and. kloroperka obturation placed coronal o the formed blood clot, Patients (a = 17) were fol Towed for various ime periods (17 days to 3.5 yeas), and then the treated tooth was extracted and the newly formed lisstes ‘were histologtealy examined. The outcomes were sims for all teeth (1) resolution of symptoms of inflammation related to forannal enlargement and oversnstrumentation in as erly a5 17 days, 2) resolution of signs and symptoms of pathosis for the necrotic cases; and, in certain cases, @) radiographic evidence of apical closure, For the histologic analysis it was abserved that there was ingrowth of connective rissue into the ‘anal space and varied levels of mineralized tissue found along the canal walls as wellas “islands” of mineralized tissue embed: dea within the newly formed tissue (Fig, 10-1) Because dental pall is type of connective tissue with a rich supply of fbro- bass, this general finding was quite promising. However, the inclusion of undesired cell ypes (eg, cementoblasts) and the lack of desired cell types (odontoblasts) inicae chat tis protocol did not lead to complete histologic regeneration of {ental pulp. Despite its shortcomings, this pioneer study nid the foundation forthe subsequent stdies inthe Geldof regen- erative endodontis In 1956, a study was published reporting that disinfection «could be established primanly with interapposntment medica- tion with pelyantbiotc mixes (ihree diferent formulations used in. fve cases)” The investigators did not purposely evoke intracanal bleeding sm his study, but instrumented ‘canals short of what they thought to be vital tissue, determined by visualization of ussite and pain sensation upon instrament tion. Signs and symptoms of disease and continued root development were resolved forall reported cases, The study represented the first reported case in which polyantibiotc pastes were used in imamate necrotic teeth for disinfection and to promote root development. Five years later, another stady was published that included the use of antibiotics in the disinfection protocol and the intentional promotion of intra ‘anal bleeding."” Resolation of symptoms and coatinted toot development was a common finding, However, the histology of the extracted teth demonstrated that connective taste was formed in 28 out of 35 teeth, whereas cellular cementum was formed in 18 out of 35 teeth. Again, these protocols generated acceptable clinical outcomes (eg., healing of apical periodon- Lis, lack of symptoms, ete.) with only partial evidence of denial pulp phenotype. Collectively, these findings laid the foundation for contemporary regenerative endadontics, dem- constrating that repair could take place following root canal ddisinfection in immavure teeth, ‘The first case report of a “contemporary” regenerative end- odontic procedure occurred in 2001." Since then, there has bbeen an exponential increase in published cases reporting unprecedented clinical outcomes such as resolution of signs and symptoms of apical periodontitis, continued root develop- ‘ment, and, in certain cases, normal nociceptive responses 10 vitality testing. Despite the lack of randomized clinical trials, these published clinical observations support the hypothesis that patients with otherwise limited treatment options benefit from these procedures, Importantly, the field of regenerative endodontics has seen a dramatic increase in knowledge gained from translational baste science studies evaluating the interplay of the tissue engineering components (stem cells, growth factors, and scaffolds) applied to the clinical need and challenges. PRECLINICAL STUDIES ON REGENERATIVE ENDODONTICS Applying the principles of tissue engineering to the develop- ‘ment of regenerative endodontic procedures requires research, fn the correct spatial assembly of distinct stem cells, growth factors/morphogens, and scaffolds to form a functional pulp- dentin complex.” "*"" In this section, we review each of these critical components in tor, Stem Cells Stem cells are defined as a distinct subpopulation of undlffer= entiated cells with self-renewal and differentiation potential They can be clasifed as pluripotent or multipotent cells. Pa ripotent stem cells have the capacity of becoming specialized ‘ells and belong to all three germ layers. Embryonic stem cells ae the best example of pluniporent cells There i a significant body of research on embryonic stem cells, but ethical, legal and medical (isue-rejection) issues can render these cll types unsuitable for clinical applications "* True pluripotent stem cells can only be found in the developing embryo, and the Iharvestng of these cells requires destruction of the embryo, Ihence the legal and ethical concerns with such practice. Dr. Yamanka and colleagues reported the groundbreaking finding that somatic cells can be transformed into pluripotent stem cells—namely, induced pluripotent stem cells GPSC)."” The use of PSCs does not have the same legal and ethical concerns as the use of embryonic stem cells, but iPSCs share the lack of control aver their uninhibited proliferation and differentiation of embryonte stem cells. These cells tend to form teratomas after implanted into a host, a ue testament of their high pro- Iierative and differentiation capacities, but this makes ther unsuitable for immediate common clinical practice”! On, the other hand, all adult mesenchymal stem cells are more restricted in their capacity to differentiate, only forming Usstes ‘of mesenchymal origin, and therefore are classified as mmult- potent” These cells can be found comparcmentalized within tissues in “stem cell niches.” The mesenchymal tissues (e g Ho CHAPTER 10 Regenerative Endodontics. 449 FIG. 10-1 Radograpic ana histologic ndings rom acetal inisor wth nacre pulp tom Nygasc- sty. ‘A Evdence of fle going beyond the apex. Theres also evidence ofa radolucenay inthe apical area, B, Secon radiogeoh at 14 monhs, taken shorty belo tooth as (acted, showing the shar fl, Hstobg section of same woth, showing fibrous connective tsue has gown into the apical 2 mm of ta tooth, D, Higher Iagniienion (per righ) shows evidence of wrat appears to be cementum cepestan on the cara wall and flrous conestve tissue Inthe pup space. E, Evidence of collagen bundles in the canal scace. rom Nygaaro-Ostoy B: he oe othe blood lot in erdodotc tery: an experimental solo stu, Acta dona! ‘rand 18323, 196!) bone, dental pulp, periodontal ligament, etc) appear to have an enriched population of adult stem cells” These cells were first found in bone marrow decades ago and were characterized as self-renewing and plastc-adherent, and they formed cell colonies with a fibroblastic appearance.” They were initially called stromal stem cells but later received the now widely accepted name mesenchymal stem cells (MSCS). Most stem cells found in the orofacial region are MSCs." Different populations of adult stem cells have been ident- fied in tissue compartments in the oral region. These include stem cells ofthe apical papilla (SCAP), inflammatory periapical progenitor cells GPAPCS), dental follicle stem cells (DFSCS), dental pulp stem cell (DPSCs), periodontal ligament stem cells (PDLSCS), bone marrow stem cells (BMSCs), tooth germ progenitor cells (TGPCS), salivary gland stem cells (SGSCs), stem cells from human exfoliated deciduous teeth (SHED), coral epithelial stem cells (OESCS), gingival-derived mesenchy- ral stem cells (GMSCs), and periosteal derived stem cells (PSCs) (Fig, 10-2). Although stem cells have been identi- fied in most oral tissues, the stem cells more likely to be involved im REPS are localized around the periapical region. ‘These include SCAP PDISCs, BMSCs, iPAPCs, and DPSCs (if vital pulp is still present apically) “The apical papilla and its residing stem cells (SCAP) were first characterized in 2006." The apical papilla (Fig. 10-3) is a dense reservotr of undifferentiated MSCs with great prolifera tive and odontogenic differentiation capacity:®”"” Importantly SCAP are regulated by Hertwigs epithelial oot sheath through, a series of complex epithelial-mesenchymal interactions that dictate root development and shape.*” Further, the close prox: inmity of the apical papilla to the apices of teeth in continwum, with the root canal space makes this rich source of stem cells. readily available for regenerative endodontic therapeutics. The IPAPCs represent another tmmportant potential source of stem. cell for regenerative endodontics in teeth with well-established. apical periodontitis." Lastly, stem cells of the periodontal 450 PARTI ‘The Advanced Science of Endodontics auscs~— FIG. 10-2 Schematic caning Mstaing pron! sources a gstatal slo cel n te ol envionment. Cel yes ink tooth germ progenitor cols (16PC3}; dal fick sten cele FSC salve glad som cals {S0SCS} som cols ofthe apical napla SCAM, drt pulp stom cal {DPSCS; ntamedperpicalprogntor cols (PAPCS stom cals fom human ‘xed dein eth SHED} periodontal igament tem cel PLSCS) tone mar sta cels BMS) an, as straedin the nse. etl slom cls (08S gra-ard mesanchymal stm eal OMS); and persteal stem cel (SCS) From Kargreaves KM, Diogenes A Tete FB: Treat options. ilo basis of regenerate endtontc procedures ‘dod 39830, 2073) ligament (PDL) and bone marrow should be also considered as stem cells sources for regenerative procedures because the ‘ction of mechanical disruption of the apical ussue (evoked bleeding) could also trigger the release of these cells, albeit their relive abundance is thought to be significantly Tess than SCAP and IPAPCs, In 2011, a study was conducted to evaluate the presence of mesenchymal stem cells following the evoked- bleeding step in regenerative procedutes.”” It was found that there is a substantial influx of mesenchymal stem cells into root canals during regenerative procedures resulting in an increase greater than 700-fold in the expression of MSC markers (Fig. 10-4). In addition, the eells could be harvested from clinical samples and examined under confocal micros- copy (Fig. 10-5), This was the first demonstration that REPS are stem cell-based therapies Although this study did not ‘evaluate whether the MSCs detected in REPS are derived from the apical papilla, it was assumed that these cells were SCAP. because the evoked bleeding step lacerated the apical papilla However, these MSCs are a heterogencous population of eels, that could come from any of the periradicula tissues after the mechanical step of evoking bleeding into the root canal system ‘The delivery of substantial concentrations of MSCs into the root canal space, despite advanced apical periodontitis or abscess, points to an impressive survival capacity of these cells Im these clinical presentations, low oxygen tension, low pH, and a high concentration of endotoxins and inflammatory mediators are expected," Indeed, the finding of high ‘concentrations of the inamune ell marker CD14 in those cl cal samples indicates that there was still a substantial chronic inflammatory exudate present at the apical region of those teeth. These findings raise the question of how MSCs such as, SCAP can survive daring apical periodontitis where a complex microflora, an array of inflammatory mediators, immune cells, and presumably low oxygen tension are commonly encoun tered, The biologic reason for this apparent resilient survival, FIG. 10-3 ACC, Dissecton of an immature permanent tooth inating the extent ofthe apical pala, Note that tis structure Is Hal facerated dung the evoked bleeding ste of revascsarzation cases and thus cols ‘rom tis stuctue, Including mesenchymal stm cols the apical papi (SCP), are lay to be delwored int ‘he root canal space. rw in C denotes junction of apa pala and dental pul. (Courtesy Dr. Michal Henry) Bl syseric loos +000 [Bi brracanal saline a e00 [Bb hrracanal ood ge ij = = 50 38 «0 Be Z £* 2 a 10 ° core eo108 FIG. 10-4 Eyoked-bleding step in endo regenerative procedures in Immature teeth with open apices ads to significant Icrese in expression of undfreatod mesenchymal str col markrs Inte root canal space Systemic bled, san rigaton and intracanal lod sarpes were cllcted uring secand vist of regenerate procedures. Realtime RT-PCR was per formed by using RNA scated from each sample as template, vith validated spac primars for target gonas ard 18S ribosomal BNA endogenous cot, Eprassin of mesere-yal tam cell markars CD73 and CD105 was upregu lola after the evoked eedng sto In regenerate procedures. Data were rnorralzd to the housekeeping gene 18S lvls and presented as mean = Standard dvition fo Ineease In eatin to systemic blood levels for aach ‘gone and aay th one-way analysis of variance wth Borfrani post rac lesl (0 = 8; "P< 05; “P< 01; ns, rot satistoaly signfean). From Lovelace TWH, Henry IA. Hergeaves KM, Diognes A: Evaluation of the olvery of mesonetymal stam calls ino te rot canal space of norte immature teth ater cliical regenerative erdooontc procedure, J Endod 37:133, 2011) despite challenging conditions, may be explained by the rela tively low density of blood vessels in the apical papilla in comparison to the adjacent dental pulp, whereas the dental follicle surrounding the apical papilla is highly vascularized and may act as a capillary bed to supply nutrients to SCAP™ Indeed, the apical papilla was found to remain vital despite complete pulpal necrosis and advanced apical periodontitis in an animal model of endodontic infection." Further, i has been demonstrated that hypoxic environments enhance the proliferation, survival, and angiogenic potential of dental stem Poo" Inerestingly, similar enhancing ellects were observed when dental stem cells were exposed to bacterial by-products such as endotoxin.” Thus, it appears that SCAP and surrounding stem cells are equipped to survive and mai tain their potential for differentiation in adverse conditions such as apical periodontitis and apical abscesses. Nonetheless, stem cells delivered into root canals after bleeding is evoked, from the periradicular tissues are likely from various apical sources oF niches The dental pulp can be viewed as a core of innervated and vascularized loose connective tissue surrounded by a Iayer of odontoblasts. The major cell type of this core region CHAPTER 10 Regenerative Endodontics. 451 is the fibroblast. Together with blood vessels, lymphatics, and neurons, this core tissue is embedded in an extraceltular matrix consisting of collagen and other fiber types (see also Chapter 12). Dental pulp stem cells (DPSCs) can be found throughout the dental pulp ut are known to accumolate in the perivascular region and the cell-rich zone of Hob adjacent to the odontoblast layer.” Thus, DPSCs from both sources are thought to be active participants in the process of eparative dentinogenesis Dental pulp stem cells are recruited to the site of injury following a gradient of chemotactic agents released by resident immune cells and fom the damaged dentin. The reparative dentin formed by these cells is distinct ftom the primary, secondary, and reactionary dentin that has heen lost"?** Ibis often called “osteodentin” when found to be disorganized, atubular, and having cellular inclusions. This process of cellular repair is enhanced by bioactive materials, (eg, MTA and Biodentine), These materials increase the inher- cent mineralizing potential ofthe dental pulp when used in both indirect and direct pulp applications." However, the process of tertiary dentinogenesis requires a vital pulp and the resolu tion of the etiology (eq, caries oF trauma). This process becomes disrupted when the pulp succumbs to injury, result- ing tn liquefaction necrosis of the dental pulp. Regeneration in this case is only possible with the recruiment or delivery of autologous stem cells to the canal space following adequate disinfection” Odontoblasts ate one of the most specialized cells of the pulp dentin complex with dentinogente, immunogenic, and possibly sensorial fanctions."*"" Odontablasts in the intact pulp-dentin complex are easily dentified based on their loca- tion and distinet morphologic characteristics (ie, colummar polarized cell body with cellular projections into the dentinal tubules). However, itis far more challenging to identify and characterize an odontoblast-ike cell, mainly because these cells lack a primary odontoblast morphology and unique rarkers that could be used for identification" Indeed, many rarkers used for the identification of odantoblastike cells are also expressed in other mineralizing cell .ypes such as osteo blasts. For example, both odontoblasis-like cells and osteo: blasts ate similar inthe formation of mineralized nodules and in the expression of several proteins such as dentin staloprotein (DSP), although DSP levels are nearly 400 times greater in odontoblasts than in osteoblasts." Measuring only one oF two characteristics of cell might not conclusively identify whether the cel is a uc odontoblast. Even among odontoblast, the phenotype varies in cells located in the apical (squamous shape) versus coronal (Call columnar) pulpal tissue. Impor- tantly, molecular studies have identified many of the genes selectively expressed in odontoblasts." Lastly, an inter- mediate lament protein called nestin has been shown to be preferentially expressed in odontoblasts or odontoblast-like cells when in the active secretory function, Nestin expression could be used in conjunction with other markers to better identify odontoblast-ike cells" This knowledge is expected to aid future studies characterizing the conditions necessary for mesenchymal cells of multiple origins to differentiate into odontoblast ike cells Its likely that definitive cellular iden- tification depends on both the morphology of the cell and an assessment of the expression of mulciple genes AC least five different types of postnatal mesenchymal stem. cells, addition to DPSCs, have heen reported to differentiate 452 PART II The Advanced Science of Endodontics cee’ corre) -anning confocal mcrosco riracanl expression ot 1G), whereas nucle appear blue as stan Diogenes A: Evaluation oth ater crial regenerate endodontic procedure, J into odontoblastlike cells, including SHED," SCAR IRAPCs,°" DFPG," and BMMSC."* One study demonstrated that overinstrumentation imto the periradicular tissues fol- lowed by bleeding into the canal space results ina robust influx of cells with mesenchymal stem cell markers in fully mature teeth, similar to that seen sn immature teeth Thus, t appears that MSCs {rom the apical region ean be delivered into the root canal spaces in both immature and mature teeth. However, there is growing evidence that MSCs have decreased prolifera tive and differentiation potential with aging Further research is required to elucidate the age limit for the use of autologous dental-derived stem cells, but these findings suggest ive procedures may be applicable co mature fully h in adults, In fact, in a proof-of-concept case report, resolution of apical periodontitis followed by narrowing of the canal space and apical elosure was seen in two fo we ceeth in adult patients treated with regenerative end odontic procedures." Thus, more research and development tained wih antooces aginst 62105, CD73, o” St C a sar yal to el maar (A) an CD10 tn TO-PRO-3. chy stem calls root canal spaces dung ragonerat ood samples afer tre evoke 0 and eva erokad-lessing step showed nn By, and STO WA, Hargtea M1) tod 37-133, 2 is required to make regenerative procedures more predictable for immature teeth, and these procedures may transition to be applicable to fully formed teeth Growth Factors/Morphogens Dentine is composed of collagen fibers (90%, collagen type 1 and noncollagenous matrix molecules (proteoglyeans, phos- phoproteins, and phospholipids). The collagen fibers act as a grid or matrix, and this structure behaves as a scaffold upon which mineralization can occur. Dentine phosphoprotein (DPP) and dentine sialoprotein (DSP) are the most abundant dentine-specific proteins among the noncollagenous proteins of organic matrix." DSP resembles other sialoproteins such as, bone sialoprotein, but its precise function is still unclear; st may have a role in matrix mineralization." Both DSP and DPP make up part of the small integrin-binding, ligand Ninked glycoproteins (SIBLINGS), which include dentine matrix acidic phosphoprotein 1 (DMP-1), bone sialoprotein, TABLE 10-1 iru) Peas RTOS Growth Factors in Dentin Matix ‘Transforming growth factor beta-1 Cassidy at al, 1997 (TGFB-1) Transforming growth factor bota-2 Cassi ot al, 1997°* rarp-2) ‘Transforming growth factor bete-2 Cassidy et al, 1997 (TGFB-3) Bone morphogenic prtuin-2 Thomadakis eta, 1999" @uP.2) Bone morphogenic protein 4 ‘Aout et al, 2000° BMP) Bone morphogenic protein -7 Tomadakis eta, 1999” @wP-7) Insulin growth factor-1 GF-) Insulin growtsfactor-2 GF) Finkleman a a, 1990" Fikleman et a, 1990" Hepatocyte growth factor (GE) Tomson et al, 2013°* Vascular endothelial growth factor Robers-Clark and Smit, (veCr, 000" ‘Adrenomedulin (ADM) Musson ota, 2010"* osteopontin, osteocalcin, and osteonectin. These proteins are nly a small par of the whole cocktail of noncollagenous pro: teins that form components ofthe dentine.” Research on dentine structure and composition has high- lighted thatthe mateix contains some components that may be Important in regulating tissue due to their bioactive properties For this reason, dentine is today considered a. reservoir of growth factors and cytokines"! These growth factors/ cytokines are secreted by the odontoblasts during primary den- Linogenesis, becoming sequestered and “fossilized” into the dentine after biomineralization (Table 10-1). However, they may become solubilized by demineralization of the matrix, bacterial aid (caries decay), chemical treatment (EDTA rinsing solution, calcium hydroxide or acid etching for bonded resto rations), or restorative materials such as mineral trioxide aggregate and Biodentine *”*" “These growth factors and their receptors have been shown to be present at the enamel organ-dental papilla interlace by immunohistochemistry and in situ hybridization during tooth development and have been implicated in odontoblast dliferentiation ‘* Growth hormone (GH) plays a paracrine or autocrine role in denzal development.” 4 IGE-L and -2 (of the family of IGF> insulin-like growth, factor)" ‘¢ TGFB-1,-2, and -3 and BMP-2, 4, and -62" play a role in the polarization and the differentiation of adontablasts» Notably in adult pulp, TGFB-1 plays an important role in the regulation of the inflammatory response and tissue regenerative processes.” The dental pulp bas well recognized regenerative potential observed in the process of reparative dentinogenesis.°”2 In CHAPTER 10 Regenerative Endodontics. 453 this process, dentin-derived growth factors are thought to play a key role to be deciphered into the regulation of progenitor cell recruitment, cell proliferation, and differentiation of new dentine-secreting cells.”?*" Indeed, the diferentiation of new odontoblas-ike cells has also been reported following pulp capping with baste fibroblast growth factor (FGF), TGF-BI,"" and BMP-7."° The sequestration of these growth factors in the dentine matrix and their subsequent “fossilization” during the miner- alization process appears key tothe pulp healing process where their release from the matrix may be responsible for various signaling events. These growth factors are extremely potent and have a variety of cell signaling properties. However, their precise localization in the dentine” and their various biologie roles remain to be elucidated, is possible to imagine opportunities for therapeutic stim- ulation, inducing targeted release of these proteins. For example, treatment of dentin with EDTA solution has been shown to dissolve the mineral phase, liberating growth factors that orchestrate the stimulation of progenitors or stem cell diferentiation "**"* Etching with orthophosphoric acid, used for conditioning the dentine in bonding procedures, also pro- rmotes demineralization of the dentine and liberation of bio- logic factors." For a long time, calcium hydroxide has been used as a protective lining, especially beneath amalgams fill- ings, or as a canal disinfection medication, It has been shown to have the ability to release bioactive components from the dentine, including growth factors.” Unlike dentin-etching acids, which only have brief contact with the dentine, eacinm hydroxide remains in place heneath restorations or in canals allowing fora gentle and continued dissolution, thus releasing growth factors, its action is prolonged and potentially contro: Iable depending on the form of the product. Lastly, calcium hydroxide, a by-product of the use of MTA and Biodentine, appears to underlie release of bioactive dentin-derived growth factors by these two bioactive materials”: Thus, clinicians may take advantage of potent growth factors stored within dentin with the use of chemical treatments and materials that promote the release of these factors. {kis also important to clearly keep in mind that a second level of regulation exists during dental development (and thus during pulp regeneration process)—transcription factors Notably, Msxl_ is expressed in. polarized. preodontoblasts, ‘whereas Msx2 is present in mature odontoblasts. Protein and transcripts for Msxl have been identified in the pulp mesen- chyme at early stages of tooth development and their concen tations decreased atthe bell stage.” The expression of these transcription factors is under the control of growth factors, and they can ultimately have broad-ranging elfects. Significant, BMP4 upregulates Msxl andl Msx2 expression. In (ura, tra scription factors regulate further growth factor expression, for example, Msx upregulates BMPS synthesis in the mesen- chyme, and Msx2 regulates Runx2 and osteocalein gene expression during edontogenesis.” Growth factors and transcription factors are central co the cascade of molecular and cellular events during tooth develop- rent and are responsible for many of the temporospatial m phologic changes observed in the developing tooth germ. For these reasons, they are also likely involved in the regeneration process 454° PART II The Advanced Science of Endodontics Itis also important to consider the nature of the signaling process between the injurious agent and the pulp cells, Bacteria and their toxins are key candidates inthe direct stimulation of pulp cells” Lipopolysaccharides (LPS) and other bacterial, toxins initiate intrapulpal inflammatory provesses by activa tion of Toll-bke receptors (¢ ., TLR¢# activation by LPS). "°°" Importantly, both progenitor and dental stem cells have been shown to express these receptors." Thus, ster cells within the dental pulp or periradicalar tissues are equipped to detect ‘microorganisms. Exposure of these cells to microbial antigens thas been shown to directly modulate the proliferation and dif- ferentiation potential of these cells "2°" Lastly, eyto- ‘nes commonly found in the inflammatory miliew Gincluding that of the dental pulp) have a profound effect on stem cells For example, it has been shown that TNF-alpha stimulates Uiflerentiation of dental pulp cells toward an odontoblastic phenotype via MAP kinase pathway activation and p38 phos- horylation.""2" Therefore, stem cell fate within the dental pulp ts ultimately dictated by a complex cascade of intracel- Tolar signaling pathways activated by agents released from, microorganisms, dentin, and immune cells Interestingly, morphogens are not only naturally occurring factors found within teeth. Several growth factors have also been evaluated for their ability to rigger the differentiation of selected mesenchymal stem cell populations into odontoblast- Wee cells (Table 10-2). Interestingly, several case studies have reported that patients taking long-term corticosteroids often present with dramatic reduction ofthe radiographic size of the pulp chamber and up to a fivefold increase in the Uhickness of the predentin layer "2" Although these were medically complex patients (¢. those experiencing renal failure) taking multiple drugs, the use of corticosteroids appeared to be associated with the observed increased activity of human odon- toblasts. Further, these unexpected “side-effects” were also observed in a retrospective study evaluating the association of pp calefcations and the long-term use of statins "” These incidental effects of commonly prescribed medications were further evaluated in translational studies that have extended this general observation by demonstrating that the application of dexamethasone or statins greatly increased the differentia tion of human dental pulp cells into odontoblast-ike cells" ‘This was particularly evident when dexamethasone was com bined with 1,25-dihydroxyvitamin Dy" Merely changing the composition of growth factors completely altered the differen- Uiation of these cells, wth the same population of cells able to ‘express markers of odontoblasts, chondrocytes, oF adipocytes, depending on their exposure (0 different combinations of srowth factors Such findings emphasize the importance of growth factors im guiding the differentiation of these cells, Other studies have evaluated growth factors administered alone or in various combinations for promoting differentiation of odontoblast-ike cells Effects of Selected Growth Factors on the Differentiation of Odontoblast-Like Cells TABLE 10-2 Growth Factors Cell Source Dexamethasone Human dental pulp Dexamethasone and Vian Ds Dsramethaone and Ascorbte- 2-phospate and FGhcerophossate Insln an ndmetiacin ad 3 ‘sobatyl-1-methybanthine (IMBX) Dexamethasone and insulin an ‘Ascortte-2-phosphae and ‘Sodium pyruvate and TGF-B1 Human dental pulp Human or rat dental pulp Human dental pup Human dental pulp Growthidierentiation factor 11 Dental pulp (carts) Simvastatin (statins) Human dental pulp LUM mineralization protein (LMP-1) Human dental pulp Bone morphogenetic proteins Dental pulp Ratimonkey dental pulp Human or rodent pulp Nerve growth factor (NGF) Immortalzed apical papilla Fbrobast growth factor 2 Human dental pulp Dentin matrix protein 1 Rat dental pulp Phenotype Condition ‘Authors COdontoblast-the in vitro x 8 wooks Muang et a, 2006" Odontoblast-tke bn vox 8 weeks Huang et a, 2006" Odontoblast-tke ia vitro> 3 wooks Wel eta, 2007" Zhang et al, 2008" Adipocyte Into x 19 days Wel eta, 2007" Chondroeyte Invitro 8 weeks Wei eta, 2007" COdontolast.the In virfin vivo 10 days Nakashima eta, 2004" Odontoblast-ike be vitovin vivo ‘Okamoto eta, 2008" Odontolast-tke ln vitor vivo Wang et al, 2007 Odontoblast-ike vitro (Chen et al, 2008" Odontoastike fn vivo Sloan etal, 1998" Odontoblast-tke be vitovin vivo Smith et al, 1990" ‘Sith et a, 2001" Teatas,2004"” Odontoblast-tke In vivo ‘any tal, 2008" Odontoblast-tke i vitro He etal, 2008" Odontolast-tke In vivo [Almushayt etal, 2006) Several of the approaches using compounds later found co have growth factor-like effects have immediate clinical impli- cations. First, i is unlikely that a single growth factor will result in maximal differentiation, so combinations of growth factors may be requited for evaluation in clinical trials, Related to this point, many of the studied growth factors (eg, dexa- rmethasone, insulin) are drugs already approved for human use in other medicalidental applications. Second, the demonstra tion that statins promote the differentiation of an odontoblast- like phenotype suggests that patients clinically taking statins may also have narrowing of the pulp chamber space, similar to the findings previously described for corticosteroids. This ‘would be an important future area of research, Third, clinicians have long used demineralized human bone to augment healing after surgieal procedures." Demineralized human bone is thought to contain a natural combination of appropriate growth factors and scaffolds, thereby providing an appropriate environment for osteoblast differentiation or function, Extend- ing this concept, several research groups have demonstrated that demineralized human dentin has significant benefit for promoting the differentiation of adontoblast-like cells. Impor- tantly, translational studies in regenerative endodontics have demonstrated that irrigation of dentin with 17%6EDTA increases the survival of stem cells" and odontoblast differentia tion," possibly due to the release of bioactive molecules, from dentin.” Collectively these findings suggest that EDTA irrigation of the dentinal walls as part of an REP could improve clinical outcomes, Scaffolds ‘An important component of tissue engineering is a physical scaffold." Tissues ate organized as three-dimensional struc tures, and appropriate scaffolding is necessary to (1) provide 4 spatially correct position of cell Iocation and (2) regulate differentiation, proliferation, or metabolism while promoting nutrient and gaseous exchanges. Extracellular matrix mole- culesare known to control the differentiation of stem cells,” and an appropriate scaffold might selectively bind and localize cells, contain growth factors," and undergo biodegradation lover time.” Thus, a scaffold is far more than a simple lattice tw contain cells, but instead can be viewed as the blueprint of the engineered tissue Scaffolds can be classified as either natural or synthete Examples of natural scaffolds include collagen, glycos aminoglycans, hyaluronic acid (HA), demineralized or native dentin. matrix,"”?"""2"*" and fibrin On the other hand, examples of synthetic scaffolds include poly-l-lactic acid (PLLA),® polyglycolic acid (PGA), polylactic-coglyeolic avid (PLGA, polyepsilon caprolactone,”" bydroxyapatite! twicalcium phosphate," bioceramics, and hydrogels such as sel-assembly peptide hydrogels." The great majority of eur- rently published regenerative endodontic procedures involve evoked bleeding and the formation of a blood clot o serve as a scaffold." Although itis relaively straightforward as it does not require ex vivo manipulation, this simplistic approach is not without challenges. The blood clot is often dificult to achieve, and it does not have many of the properties of the ‘deal seaffold, These properties include easy delivery, adequate sechanical properties, controllable biodegradation, and incor- poration of growth factors." In addition, the blood clot con- tains a great number of hematopoietic cells that eventually undergo cell death, releasing their toxic intracellular enzymes CHAPTER 10 Regenerative Endodontics. 455 nwo the microenvironment, which may be detrimental to stem cell survival ‘Another approach for creating a scaffold involves the use of aurologous plateletrich plasma (PRP). It requires minimal ex vivo maniptlation, being fairly easy to prepare in a dental setting, PRP is rich in growth factors, degrades over time, and forms a three-dimensional fibrin matrix." Platelet rich fibrin (PRF) is an alternative to PRE as it has a three dimensional architecture conducive with stem cell proifera- tion and differentiation and contains bioactive molecules *” ‘These autologous scaffolds have been used successfully in regenerative cases." "*"" However, it should be emphasized that, despite their reported use, there are some drawbacks to their clinical use: the process requires collection of intravenous blood that can be challenging in children, the diversity and concentration of growth factors within PRP and PRF prepara tions are not contoliable:* "and they Tack temporal deg radation control and the mechanical strength to support a coronal restoration. Thus, despite some desirable characteris- tics, other scaffold alternatives to PRP and PRF should be carefully considered. Hydrogels are a class of scaffolds composed of three dimensional hydrophilic polymers that absorb water or tissue fluids up to several times their weight." These water-swollen materials are easily injectable in their colloidal form, undergo- ing gelation by chemical (¢., changes in pH and osmolarity) or physical eg., temperature change) cues. These materials are highly tunable, biocompatible, and can be designed to resemble naturally occurring extracellular matrices.“ They are of particular interest for regenerative endodontics because they can be easily injected into narrow root canal spaces andl can be ‘modified to deliver chemotactic and angiogenic agents to drive stem cell homing and supportive angiogenesis. Hydrogels rade of sel-assembly peptides (eg, Puramatrix)” show great potential to be used in endodontic issue engineering because their sequence includes short peptide sequences, similar to those naturally occurring in tissues, enhancing cell attachment and proliferation Delivery System Even with selection of the appropriate cell source, growth factors, and scaffold, the resultant mixture must be delivered in a spatially appropriate fashion into the space of the root canal system. For example, nearly all cells of the body are within 0.1 t0 1 mm of a blood vessel in order to maintain sedequate diffusion of oxygen and nutrients” This represents a challenge still to be overcome in the currently performed regenerative endodontic procedures that recruit stem cells"? to a canal space devoid of lateral vascularity and several millime- ters away from apical blood vessels, If one were to inyect cells, sma cell-based approach, along the entie coronal-apical extent cof a root canal system, the majority of cells would be expected tw succumb to tissue hypoxia, Interestingly. it has been dem- onstrated that under hypoxic conditions, stem cells proliferate faster and release greater levels of angiogenic factors such as vascular endothelial growth factor | (VEGF) that promote targeted angiogenesis into the engineered space.” Thus, an alernative approach would be to inject a scaffold with chemo- tactic factors into the root canal. This approach is called cell hhoming, as cells are atracted to the scaffold along with sup- portve blood vessels in a progressive manner”, instead of being abruptly delivered to an avascular space Ge, similar to 456 PARTI ‘The Advanced Science of Endodontics lusting boengin vith a nora pulp and apial asin & seatfla of apical stern oa in rity wth vascular su arreaves KM: An update on cna raonerat the current revascularization procedures), the cell-homing approach can be applied in cell-tee™ (no cells implanted along the chemotactic factors, see Fig, 10-0) or cell-based approach (cells are delivered in the chemotactc-containing scaffold)” Because dental pulp can be approximated a5 a loose connective tissue core surrounded by a layer of odonto- blasts, the spatial arrangement of cells and growth factors within the scaffold may be particularly important t9 promote odontogencsis without having complete calcification of the root canal system, Complete recapitulation of the pulp-dentin complex architecture requires additional research efor. Translational Studies Several elegant studies in regenerative endodontics have used. various translational methodologies including evaluation of clinical samples,"” organotypic root canal models,""””” tooth slice models," “whole tooth culture, and animal models"! These studies have been crucial 10 provide a strong scientific foundation for the field of regenera tive endodontics while allowing for clinical treatment protocol optimization and the development of new treatment strategies such as inclusion of scaffold and growth factors in regenerative procedures." 10 js dsocto oat 8 sand charac fat the cara space panel C)katng tothe populaton ofthe canal space wth stem cols 80% of Ca(OH, was removed, wih more ecient removal carats inigated wt PUL (8). Data removal standard error of the mean, *P-< 05 armision rom Bardot JA, Chen PB, Teva FB, ctferentirgation procedures, J Endod 40:1172, 2014) EDTA and saline using sod by the Student f test (0 iaganos & Evaluation of trpleantbate paste remaval by CHAPTER 10 Regenerative Endodontics 459. 1000-fold drop in bacteria, These findings were confirmed in. another related dog study." This study provides strong support for the eflectiveness of triple antibiotic paste in disinfection of immature teeth with apical periodontis As stated previously, calcium hydroxide has been the second nost used intracanal medicament in published cases. This application represents a new use ofa long established sntraca- nal medicament n endodontcs, Although Ca(OH), appears to be less effective against some intracanal bacterial spectes ths antibiotic paste formulations,” its use is associated with lower cytotoxicity to stem cells," release of important bioactive sgrowih factors from the treated dentin,” and greater survival and proliferation of stem cells in the presence of the condl tioned dentin." Also, the relatively short-term use of this medicament in regenerative procedures does not appear sue ficient to reduce fracture resistance.** Another factor to con- sider when choosing an intracanal medicament is the ability c© remove the medicament from the canal space, One study that addressed this question incorporated radioactive tracers sn both calcium hydroxide paste (Utracal, Ultradent, Inc.) and. twiple antibiotic paste (Champs Pharmacy, San Antonio, TX)" The radiolabeled medicaments were placed in extracted teeth with standardized root canals. After 28 days of incubation, ceanal spaces were irigated with a standardized protocol using, different techniques. Surprisingly, greater than 80% of the triple antibiotic paste could not be removed from the tooth, (Fig. 10-8), and at was found not im the canal lumen, but greater than 350 jm into the dentinal tubules. In contrast, areater than 80% of calcium hydroxide was removed (see Fig. 10-8) with the remaining medicament present superficially within dentin." This isan important finding, given that drags 100: 501 °. B PP PUL « elinates ater andodonti niga, Pacolaboled root sogmets and inebated for 28 days at 37° C vith & her postive presu vation of iigants (PUB, Thee was no dit tol 20% ofthe mediarnat being le (Modted th mean percerlage of tla redo 2g 460 PART The Advanced Science of Endodontics = a4 > s000. conrl jhe Oh CHO} TAP _ DAP 100m Toi FIG. 10-9 Dentin canatoning fr 7 days wth mesdeaments used in REPS tas a profound eflect on SCAP survival. Standarded denn disks were ‘rete for 7 das vith TAP or double anibiot paste (DAP) concerrations of 1000 mgm or 1 mail), CafOk,futvacab, or stor salina (conto. SOAP in a Matiga safc (BD Bos NA) was seeded into ‘he umn of the dks atte the medicaments were removed and cuted for 7 days, Call vial (univ was determined using a luminescent assay ‘SCAP cuture on dentin teatad witr TAP or ORP atthe conceivaton of 1000 mgm. rsutzs in o vial cls Conversely, dentin can ciference rm urieat ‘ion wore detacad in tha in sks convo, Greater survival and protera- ‘weated win Ca(OH, Data are presantes as mean + slandard davation of clave Limnescance unts (n= 12/grOup "P<.001, ns, no sass diferenoe as tested by 1-may analysis of variance, From Attumaly Rl, Tewera FB, Diogenes A: Etet of

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