REVIEW : an introduction for the endodontist

P. G. S a t c h e ll 1,J.L.Gutmann2 & D. E. Witherspoon3 1Private Practice Limited to , Houston, 2Private Practice Limited to Endodontics, Dallas, and 3Private Practice Limited to Endodontics, Plano,Texas, USA

immunological response and in£ammation, and in Abstract and certain pharmacological e¡ects. Satchell PG, Gutmann JL, Witherspoon DE. Apoptosis: The understanding of the ability of clinical materials to an introduction for the endodontist. International Endodontic Journal, induce or inhibitapoptosis and the investigationof apop- 36, 237^245, 2003. tosis as it relates to the of pulpaland perira- dicular may eventually lead to new Apoptosis plays an important role in many aspects of treatment approaches for the endodontist. The purpose endodontics, yet there is a paucity of information in this of this review is to familiarize the clinical endodontist regard in the endodontic literature. Apoptosis is a single withcurrent knowledge on apoptosis as it relates to the deletion of scattered cells by fragmentation into mem- pulp and periradicular tissues. brane-bound particles that are phagocytosed by other cells. It is a key process inthe embryological development Keywords: apoptosis. of the , periodontal ligament and supporting oral in the progression of oral , bone resorption, Received 21May 2001;accepted 2 January 2003

Introduction Why and how is apoptosis important to endodontics? Apoptosisisessentiallycellsuicide,andwas¢rstdescribed Apoptosis plays a ubiquitous role in the body. Amongst in the BritishJournal of Cancer i n 1972 (Ke r r et al.1972). other things, apoptosis is a key process in oral develop- According to these authors, at the appropriate time ment (Vaahtokari et al.1996,Haradaet al. 1998, Cerri and under certain conditions, cells self-destruct without et al. 2000), the progression of oral disease (Polverini & damaging adjacent cells. This process is occurring con- Nor 1999), periradicular lesion development (Onishi stantly and can happen to seemingly healthy cells. et al.1997), resorption (Hughes & Boyce1997), immuno- Whilst it is unique to animals and is studied classically logical response and in£ammation (Onishi et al.1997), in the nematode worm Caenorhabditis elegans, it hasbeen wound healing (Fanning et al.1999) and in certain phar- increasingly studied in vitro and in higher organisms. macological e¡ects (Hughes et al.1995).The understand- There has been an explosion in the number of articles ing of the ability of clinical materials to selectively on the subject of apoptosis. A conservative estimate induce or inhibit apoptosis is leading to new treatment places the number at nearly 45 000. Whilst apoptosis modalities (Bamford et al. 2000). An understanding of has been a topic of extensive research in the scienti¢c the mechanisms of apoptosis may lead to adjunctive community for some time, it has received little attention treatments for pulpal and periradicular in the endodontic literature. The purpose of this article through yet unexplored pathways. is to familiarise the endodontist with apoptosis and to discuss some of the potential implications of apoptosis What is the apoptotic process? to endodontics. The entire process of apoptosis takes about 1 h from initiation (Figs 1and 2). The initiating triggers are many Correspondence: Dr James L. Gutmann, 1416 Spenwick Terrace, Dallas, Texas, T X 75204 -5529, USA (Tel.: þ1 214 827 5378; fax: þ1 214 827 4848; and varied, and are grouped broadly as physiological or e-mail: [email protected]). nonphysiological. These include, but are not limited to,

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36, 237^245, 2003 237 Apoptosis Satchell et al.

Figure 1 The major stages of apoptosis include the initiating trigger (1) that leads to the activation of the intracellular mechanism for the apoptotic process (2). Morphological changes of the (3) include cell membrane changes that signal phagocytic cell recognition and elimination (4) without promoting in£ammation.

the following: Fas ligands (Fas), tumour factor as the nuclear membrane (Nicholson & Thornberry (TNF), nerve growthfactor (NGF), nitric oxide (NO), lipo- 1997). The cell shrinks in the process and there is a loss polysaccharide (LPS), host immune reactions, kinins of cell^cell junctions resulting in detachment from adja- and glucocorticoids (McKenna et al.1998). The best char- cent cells. The chromatin condenses, the cytoplasm acterised apoptotic trigger is the Fas ligand, a member ‘blebs’ (forms so-called‘pseudopods’) and the cell breaks of theTNF super-family.The Fas receptor is a cell surface up into fragments known as‘apoptotic bodies’.Indirectly glycoprotein that mediates apoptotic signals from the activated endonucleases lead to breakdown of the DNA cell surface into the cytoplasm (Yoshioka et al. 1996). (Kerr et al.1972) into multiples of180^200 base pair frag- When the Fas ligand binds to the Fas receptor on the cell ments (Cohen & Duke1984). Finally,either membrane, the newly formed Fas complex is allowed to or adjacent cells phagocytose the apoptotic bodies (Kerr associate with intracellular proteins.The morphological et al.1972) (Figs 1and 2). changes of speci¢c intracellular proteins induced by this complex result inthe activationof other substances such How does necrosis compare to apoptosis? as IL-1bconverting enzyme (ICE). In this particular mechanism, and there are many, Cells usually die either by necrosis or apoptosis. The Procaspase (inactive form) is activated to the protease characteristics of apoptotic death are more clearly Caspase. An ampli¢cation cascade then ensues with understood when compared to the characteristics of Caspases activating other Caspases, eventually cleaving necrotic death(Table 1). Necrosis is a pathological death the host cell byacting on avariety of cell structures such of cells resulting from irreversible damage that occurs

238 InternationalEndodontic Journal, 36, 237^245, 2003 ß 2003 Blackwell Publishing Ltd Satchell et al. Apoptosis

Figure 2 Various initiating triggers e¡ect the activationof a central apoptotic signal.This central signal that can be blocked by bcl-2 leads to the activation of proteolytic enzymes via the caspase cascade.Those enzymes lead to morphological changes in the cell structures including the nucleus, cytoskeleton and membrane. Cell membrane changes include expression of proteins for recognition by phagocytic cells. inthe dental pulp and is aterm commonly used in pulpal deposits. After such changes, the outlines of individual diagnosis. The earliest irreversible changes are mito- cells are indistinct and a¡ected cells may become chondrial, consisting of swelling and granular calcium merged, sometimes forminga focus of coarsely granular, amorphous or hyaline material (Stedman 1995). These Table 1 Comparison of classic features of apoptosis features include cell swelling, membrane lysis and an (programmed ) and necrosis (pathologic cell death) in£ammatory response (Wyllie et al. 1980), and are dis- (Polverini & Nor1999) tinctly di¡erent from the features of apoptosis described Apoptosis Necrosis above. Controlled process Uncontrolled process Energy dependent (ATP required) No energy required (passive) Cells shrink (apoptotic bodies) Cells swell (lysis) What role does apoptosis play in development? No loss of membrane integrity Membrane integrity lost Noninflammatory Inflammatory Apoptosis plays an important role in all stages of life. No scarring occurs Scarring Developing human branchial arches, embryonic tails Individual or small cell groups Large cell groups (organ segments) and ¢nger webbing cannot resorb before birthwithout Nuclear fragmentation Nuclear dissolution organised . In mature organisms, Physiological stimuli Pathologic stimuli homeostasis is maintained by balancing the continuous

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36, 237^245, 2003 239 Apoptosis Satchell et al.

mitosisanddi¡erentiationofcells withtheapoptoticpro- formation by odontoblastic layering rather than by cell cess (Kerr et al. 1972). death. Apoptosis is more evident in after Apoptosis has multiple roles in tooth development injury to odontoblastic processes as seenwithcavitypre- from the beginning of tooth formation to the completion paration (Goldberg et al.1994, Bronckers et al.1996, Kita- of root development. There is evidence of apoptosis in mura et al. 2001). There is evidence that reparative the reduction of cells of the stellate reticulum, at the (tertiary) dentine formation, in response to injury, is initiation of enamel formation (Vaahtokari et al.1996, associated witha large decrease in thenumber of odon- Baratella et al. 1999) and in the stratum intermedium toblasts. Up to half of the pulp odontoblasts can be elimi- (Bronckers et al.1996,Vaahtokariet al.1996). This process nated in only 4 years by this process (Franquin et al. also occurs during the transition stage between secre- 1998). and vascular endothelial cells of the tion and maturation of ameloblasts during enamel for- pulp proper also show evidence of apoptosis (Franquin mation (Nishikawa & Sasaki 1995). After enamel et al.1998). Although certain characteristics of apoptotic formation, approximately 25% of the ameloblasts die deathare favourable to necrosis (i.e. an in£ammatory and following enamel matrix maturation, another 25% response is not triggered), apoptosis does not lead to pulp undergoapoptosis (Joseph etal.1994). Apoptosis has been recovery. This may be due in part to the elimination of shownto occuraround the crowns of teeth during tooth regenerative cells byapoptosis (Goldberg et al.1994).Ulti- eruption (Bronckers et al.1996, Kaneko et al.1997), play- mately, when apoptosis occurs, apoptotic bodies and inga major role inthe eliminationof reducedameloblasts debris are phagocytosed by major histocompatibility located at cusps (Shibata et al. 1995). It is thought that Class II (MHC II) positive and negative macrophages the inhibition of apoptosis in reduced ameloblasts may (Nishikawa & Sasaki 1999). occur after the elimination of the tooth organ, allowing for the establishment of the junctional epithelium (Shi- What role does apoptosis play in other oral tissues? bata et al.1995).Whilst the precise mechanisms for these events remain largely undiscovered, some work has Apoptosis seems to be necessary for maintaining home- shown that bcl-2, an apoptotic inhibitor, is involved in ostasis within continually renewing tissues such as the maintaining the viability of the during oral mucosaand skin (Funato et al.1999). Gingival tissue toothdevelopment (Slootweg & de Weger1994). has a high cell turnover, and apoptosis has been demon- -like cells of the periodontal ligament (PDL) strated to occur in this tissue in 90% of the individuals exhibit apoptosis during tooth development (Cerri et al. tested (Yoshioka et al. 1996). Here, as in other parts of 2000), and apoptotic cells, probably osteoclasts, are the body, apoptosis is essentially a counterbalance to also found on the surfaces of developing alveolar bone mitosis. Unfortunately,the role of apoptosis in the di¡er- (Vaahtokari et al. 1996). Apoptosis is also responsible entiation of oral epithelial cells is not clear (Harada for at least partial elimination of the cells of Hertwig’s et al. 1998), and more work is required to fully describe epithelial root sheath (HERS) after root formation is the events associatedwiththis process in normaloraltis- complete (Kaneko et al. 1999, Cerri et al. 2000). Further- sues as a baseline to further study. more, cell rests of Malassez, which can proliferate to pro- duce periradicular cysts, may be partially as a result of What role does apoptosis play in bone? incomplete apoptosis of HERS. In the process of bone remodelling, some osteoblasts die via apoptotic mechanisms, whilst those remaining What role does apoptosis play in the dental pulp? become embedded as osteocytes (Ihbe et al.1998,Jilka Apoptosis is a part of normal pulp homeostasis et al. 1998). Many osteoclasts that lose their attachment (Nishikawa & Sasaki 1999), occurring more in the occlu- to bone die by the process of apoptosis (Hughes & Boyce sal (incisal) than in the apical portions of the pulp 1997). In this regard, a third term in addition to necrosis (Vermelin et al. 1996, Nishikawa & Sasaki 1999). Most andapoptosisis oncosis, whichdescribes celldeath asso- apoptotic cells in normalpulp can be foundat theperiph- ciated withslow ischaemiaand cell swelling.This occurs eryand are usuallyassociated withthesubodontoblastic to some osteoblasts during transition to osteocytes region rather than with the odontoblastic layer (Darzynkiewicz & Traganos1998). (Vermelin et al. 1996, Piattelli et al. 2001). Odontoblasts TNF-a has been shown to enhance osteoblast apopto- seem to compensate for the reduction in pulp chamber sis and may contribute to bone loss associated with volume as a result of physiological (secondary) dentine in£ammation (Hill et al. 1997, Ji l ka et al. 1998, Tsuboi

240 InternationalEndodontic Journal, 36, 237^245, 2003 ß 2003 Blackwell Publishing Ltd Satchell et al. Apoptosis et al. 1999). Neutrophils can also induce apoptosis of response (Onishi et al. 1997). Apoptosis of neutrophils osteoblasts, demonstrating the relationship between is the major means of ending neutrophil-associated in£ammatory cells and bone resorption associated with in£ammation (Marshall & Watson 1997). Induction of in£ammation (Kawakami et al.1997).Ontheotherhand, this process therefore could be a way of minimizing growthfactors, cytokines and other bone-stimulating in£ammation that would occur as a result of neutrophil hormones can reduceapoptosis ofosteoblasts duringper- necrosis (Onishi et al. 1997), which is mediated and iods of bone resorption (Hill etal.1997,Jilkaetal.1998). induced by certain environmental signals (Marshall & Apoptosis occurs in periradicular tissue during bone Watson 1997). There is also evidence that the failure of remodelling associated withorthodontic toothmove- apoptotic mechanisms in neutrophils can contribute to ment (Rana et al. 2001). Certain mediators may limit pathogenesis of disease (Marshall & Watson1997). Neu- the resorptive process and aid in bone formation during trophils in exudate show delayed apoptosis and loss of remodelling (Hilletal.1997).Thereis signi¢cant evidence TNF-a, which is necessary for neutrophil survival in that osteoblasts may be involved in the regulation of the extravascular £uid (Seely et al. 1998). In addition, osteoclast apoptosis (Fuller et al. 1993; 1998, Green¢eld neutrophils in diabetics donot undergolipopolysacchar- et al. 1999). In fact, most of the mediators that stimulate ide (LPS)-induced inhibition of apoptosis. This may con- osteoclast activity seem to act through osteoblasts tribute to the increased susceptibility to (Green¢eld et al. 1999). In general, however, factors that observed in diabetic patients (Tennenberg et al. 1999). stimulate bone resorption inhibit osteoclast apoptosis Calcium ions have been showntoa¡ect neutrophil apop- and factors that inhibit bone resorption promote osteo- tosis (Onishi et al.1997).The signalling mechanisms for clast apoptosis (Hughes & Boyce1997). other immune cells have been studied less extensively Apoptosis of osteoclast precursors may be one way than those for neutrophils. that the osteoclast cell population is controlled (Hughes & Boyce1997), e¡ectively reducing bone resorption (van’t What is the role of apoptosis in oral disease? Hof & Ralston 1997). Furthermore, members of the TNF super-family inhibit osteoclast apoptosis (Fuller et al. Apoptosis is widely involved in disease mechanisms of 1998) and induce precursor maturation to mature func- the oral cavity. Oral diseases in which apoptosis plays a tioning osteoclasts (Fuller et al.1998), thereby contribut- role include (Dekker et al.1997, Bloor et al. ing to bone loss. 1999), odontogenic keratocysts (Muraki et al.1997),leu- In the presence of high extracellular calcium concen- coplakia (Muraki et al. 1997), squamous cell carcinoma trations as a result of ongoing resorptive processes, (Muraki et al.1997, Kaur & Ralhan 2000, Ravindranath osteoclast apoptosis is induced (Lorget et al.2000). Simi- et al.2000), oral lymphoma (Regezi et al.1998), aphthous larly, oestrogen, glucocorticoids, bisphosphonates and ulceration (Honma et al. 1985), erythema multiforme TGF-b have been shown to stimulate apoptosis of osteo- (Chrysomali et al.1997),Sjo« gren’s syndrome (Ishimaru clasts (Green¢eld et al. 1999). et al. 2000) and mucocutaneous candidiasis (Heiden- reich et al. 1996).

What role does apoptosis play in immune cells? Can microorganisms induce or inhibit apoptosis? Apoptosis is associated withthemaintenance of immune cell homeostasis (Usherwood et al. 1999). Neu- Bacterial proteases have been implicated as inducers of trophil production is balanced by apoptosis and clear- host cell apoptosis (Wang et al. 1998). Fusobacterium ance from tissues without inducing an in£ammatory nucleatum induces apoptosis in peripheral blood mono- response (Onishi et al.1997). Apoptosis also plays a criti- nuclear cells and neutrophils (Jewett et al. 2000). This cal role in eliminating harmful or injured cells from tis- is signi¢cant because premature immune cell apoptosis sues. This suggests its participation in in£ammatory may decrease host resistance and promote infection processes and in the resolution of in£ammatory reac- (Geatch et al. 1999). tions (Onishi etal.1997). Inperiradicularlesions, apopto- Furthermore, apoptosis of both neutrophils and sis occurs predominantly in neutrophils (Takahashi monocytes decreases in the presence of LPS (Mangan et al.1999), a process that may be defective in abscess for- et al. 1993, Preshaw et al. 1999,Tennenberg et al. 1999), mation as a result of the acidic environment (Onishi resulting in a heightened immune response (Hiroi et al. et al. 1997). This lack of clearance of apoptotic cells can 1998). Another organism, Prevotella intermedia, does present a persistent antigen, inducing an autoimmune not induce apoptosis in cultured human osteoblastic

ß 2003 Blackwell Publishing Ltd International Endodontic Journal, 36, 237^245, 2003 241 Apoptosis Satchell et al.

cells (Morimoto et al. 1999), whilst in Streptococcus lation of in£ammation in a wound may in fact be derived mutans, lipoteichoic acid may be a factor in as from the epithelium because it appears concurrently there is evidence that it causes apoptosis in human den- with re-epithelialisation of the wound (Brown et al. tal pulp cells of (Wang et al. 2001). 1997, L e ona rd i et al. 2001). Generally,these interactions are complexandunpredict- Granulation tissue ¢broblasts (myo¢broblasts) play a able as microorganisms may increase apoptosis in some role in wound contraction. When granulation tissue host cells and decrease apoptosis in others. evolves into a scar, myo¢broblasts disappear, probably as Whilst bacteria and their by-products can induce pul- a result of apoptosis. Myo¢broblasts persist in excessive pal in£ammation, they do not always lead to pulp necro- scarring conditions (Desmouliere1995), possibly because sis (Bergenholtz 2000). Bacterial proteases have been of an inappropriate inhibition of apoptosis in these cells. implicated as inducers of host cell apoptosis (Wang et al. Certain mediators may be potential stimulators of apop- 1998). Although certain characteristics of apoptotic tosis in myo¢broblasts after re-epithelialization in the deathare favourable to necrosis, apoptosis does not lead palatal wound healing process (Funato et al. 1999). to pulpal regeneration (Goldberg et al.1994). What is the role of apoptosis in How is apoptosis involved in ? pharmacotherapeutics?

There is a considerable amount of research on the role of The principle therapeutic goal of apoptosis research is apoptosis in periodontal disease. Apoptosis occurs in the understanding of how to induce or inhibit apoptosis cells of the periodontium as part of normal turnover in speci¢c cells. This is probably the best approach for and remodelling (Koulouri et al. 1999), and may be even altering rates of apoptosis in target cells whilst avoid- moreprevalentthan necrosis inperiodontaldisease (Sor- ing systemic toxic e¡ects that may be otherwise asso- kin & Niederman1998). It seems to play a role in age reg- ciated. Aspirin, nonsteroidal anti-in£ammatory drugs ulation of some immune cells and may be involved in (NSAIDS), and selective cyclooxygenase 2 (COX2)inhibi- the maintenance of local immune homeostasis in tors induceapoptosis incertaincells viaacaspaseactiva- in£amed gingival tissue (Tonetti et al.1998). There is also tion mechanism. Certain drug combinations may evidence that increased in£ammation is associated with induce apoptosis by a mechanism involving the disrup- increased epithelial cell apoptosis in the periodontium tion of the cell cycle (Bamford et al. 2000). of patients withperiodontal disease (Carro et al.1997). The inductionofosteoclastapoptosis for thetreatment Various microorganisms associated withperiodontal of bone resorption is another therapy that utilizes apop- diseases have been shown to generate di¡erent short- totic mechanisms (Hughes & Boyce1997).Antiresorptive chain carboxylic acids as metabolic by-products, which drugs such as the bisphosphonate family have been canpromote in£ammation by inhibiting normal apopto- shown to inhibit bone resorption by inducing osteoclast sis of certain in£ammatory cells (Niederman et al. (Hughes et al.1995,Rogerset al.1996, Hiroi-Furuya et al. 1997). For example, lactic acid and propionic acid com- 1999) and apoptosis (Rogers et al. 1996). monly produced by oral microorganisms in periodontal Bisphosphonates inhibit osteocyte and osteoblast apop- diseases have been shown to inhibit apoptosis of various tosis contributing to their antiresorptive e¡ects (Plotkin in£ammatory cells (Niederman et al.1997,Yamamoto et al. 1999). Tetracycline can also inhibit resorption by et al. 1997, Sorki n & Nie de r ma n 1998). The resu lta nt pre - inducing osteoclast apoptosis (Cillari et al. 1998, Vernillo sence of increased numbers of in£ammatorycells results & Rifkin1998).This is part of the process by which doxy- in an elevated in£ammatory state. cycline downregulates the in£ammatory process (Liu et al. 1999). There is some thought that tetracycline uses a unique mechanism to induce this selective apoptosis What is the relationship between gingival trauma, (Bettany & Wolowacz 1998); however, details of this wound healing and apoptosis? mechanism have not been fully described. There is a strong evidence that trauma, including surgi- cal trauma, inhibits immune cell apoptosis (Ertel et al. Which dental materials have apoptotic e¡ects? 1999, Fanning et al. 1999, Ogura et al. 1999, Nolan et al. 2000). Apoptosis occurs in cells at the advancing epithe- An understanding of the di¡erence between necrotic lial edge in wound healing (Brown et al.1997).Thereis and apoptotic forms of cell deathis important for under- speculation that the signal for apoptosis and downregu- standing the pulp reaction to dental materials (Goldberg

242 InternationalEndodontic Journal, 36, 237^245, 2003 ß 2003 Blackwell Publishing Ltd Satchell et al. Apoptosis etal.1994). Inculturedcells derived fromthehuman PDL, Brown DL, Kao WW, Greenhalgh DG (1997) Apoptosis down- evidence of apoptosis in response to di¡erent dental regulates in£ammation under the advancing epithelial materials has been described (Adams et al. 1995) and wound edge: delayed patterns in diabetes and improvement may occur in damaged areas of the pulp because of cer- withtopical growthfactors. Surgery121,372^80. Carro OM, Evans SA, Leone CW (1997) E¡ect of in£ammationon tain toxic substances in those materials (Goldberg et al. the proliferation of human gingival epithelial cells in vitro. 1994). In some cases, dental materials may lead to Journal of Periodontology 68,1070^5. uncontrolled development of apoptosis within central Cerri PS, Freymuller E, Katchburian E (2000) Apoptosis in the pulp cells, but may leave the odontoblasts more or less early developing periodontium of rat molars. Anatomical intact (Goldberg et al.1994). Record 258,136^44. More speci¢cally,and related to endodontic treatment, Chrysomali E,Lozada-Nur F,Dekker NP,Papanicolaou SI, Regezi in vitro apoptotic changes are documented in human JA (1997) Apoptosis in oral erythema multiforme. Oral PDL cells exposed to calcium hydroxide (Adams et al. Surgery, Oral , Oral Pathology, Oral Radiology and 1995). Calcium hydroxide has antibacterial properties Endodontics 83,272^80. and has shown predictable healing and hard tissue for- Cillari E, Milano S, D’Agostino Pet al. (1998) Modulation of nitric mation when used in cavity preparations (Bergenholtz oxide production by tetracyclines and chemically modi¢ed tetracyclines. Advances in Dental Research12, 126^30. 2000). Calcium ions mayalsoa¡ect neutrophil apoptosis CohenJJ,Duke RC (1984) Glucocorticoid activationof a calcium- (Onishi et al.1997). Unfortunately, neither the apoptotic dependent endonuclease in thymocyte nuclei leads to cell e¡ects of composite resin on the dental pulp nor the death. Journal of Immunology132,38^42. potential apoptotic e¡ects of extruded sealers Darzynkiewicz Z,Traganos F (1998) Measurement of apoptosis. on the cells that make up the supporting periradicular In: Scheper T, ed. Advances in Biochemical Engineering/ tissues have been fully documented. Therefore, the Biotechnology. 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