Understanding pulp biology for routine clinical practiceResults: Novel biotechnological insights...

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

ENDO (Lond Engl) 2009;3(3):171–184

� 171REVIEW

Aim: To review the latest developments in the field of pulp biology, particularly those elements of spe-cific interest to clinical dentists, whilst highlighting the importance of maintaining pulp vitality for con-servative dentistry. Pulp biology is crucial to everyday practice in dentistry and the knowledge acquired,especially in the last five years on the pulp healing process, has highlighted simple but effective appli-cations. However, difficulties in communication between biologists and clinicians, mostly due to thecomplexity of biology as a discipline, are a significant obstacle to therapeutic developments and theirapplication on a larger scale. Methods: A literature review was undertaken on the current understanding of the biology of the den-tine-pulp complex, especially in the context of conservative dentistry. Results: Novel biotechnological insights have recently been discovered, including the presence of stemcell-like cells within the tooth and their potential roles in reparative and regenerative processes. Agreater understanding is also developing regarding the structure of the dentine-pulp complex, bothmacroscopically and microscopically, which may have important consequences for therapeutic choices. Conclusions: The emergence of new adhesive systems, together with disinfecting molecules, repre-sent a first step towards the application of new biological approaches to the treatment of pulpal dis-ease. Improved understanding of the many pathophysiological processes of the dentine-pulp complexand the development of new materials, which are being adapted to clinical conditions, has led to sig-nificant advances for the therapeutic principles underpinning conservative dentistry.

Stéphane Simon, Paul Cooper, Ariane Berdal, Philip Lumley, Phillip Tomson, Anthony J Smith

Understanding pulp biology for routine clinicalpractice

pulp biology, pulp regeneration, growth factors, reactionary dentinogenesis, TGFβ1Key words

Stéphane Simon,DCD, MScAssociate Researcher,INSERM, UMR S 872,Centre de Recherche desCordeliers, Paris, FranceSchool of Dentistry,University of Birmingham,Birmingham, UK

Paul Cooper, MSc, PhDSenior Lecturer in OralBiology, School of Dentistry,University of BirminghamBirmingham, UK

Philip Lumley, MSc, PhDProfessor of Endodontology,School of Dentistry,University of BirminghamBirmingham, UK

Ariane Berdal, PhDProfesseur des Universitésde Biologie Orale,INSERM, UMR S 872,Centre de Recherche desCordeliers, Paris, France

Phillip TomsonClinical Lecturer andSpecialist Registrar inRestorative Dentistry,School of Dentistry,University of BirminghamBirmingham, UK

Anthony J Smith,MSc, PhDProfessor of Oral Biology,School of Dentistry,University of BirminghamBirmingham, UK

Correspondence to:Stéphane SimonLaboratoire de Physiopatho-logie Orale Moléculaire, INSERM, UMR S 872,Escalier B, 15-21 rue del’Ecole de Médecine,75006 Paris, FranceTel: +33 970 40 58 87Fax: +33 956 83 36 93Email: [email protected]

� Introduction

Significant progress in the field of carious diseasemanagement has led to much research into themineralisation of teeth and the biological behav-

iour of the dentine-pulp complex. It appears thatthe dentine-pulp complex is able to adapt to amultitude of stimuli, invoking defence responsesto maintain pulp vitality. The main role of thepulp is to secrete dentine. When tooth develop-

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

ment is completed, the pulp maintains the den-tine through homeostatic and self-protectivemechanisms. The dental pulp is able to reinitiatedentinogenesis at any time to protect itself fromexternal injuries. However, it appears thatadvances in understanding the basic biology ofthis tissue have not yet been fully translated intobetter treatments in day-to-day dental practicebeyond minor carious lesions. When caries hasreached the dentine, it is generally assumed thatit is too late to use such techniques to intervene.When the caries extends to the pulp, a pulpec-tomy is usually considered in order to preventpainful, infectious complications. However, noguidelines clearly define the indications forpulpectomy versus pulp vitality conservation.

Many researchers have been investigating thepulp healing process for several years, and recentadvances in biotechnology have provided oppor-tunities for the development of applications inpulp vitality maintenance, reactive dentinogene-sis or revascularisation of the infected canal.

The aim of this paper is to present the relevantbasic elements of pulp biology and highlight theexisting and potential applications of biotechnol-ogy in the context of routine conservative den-tistry.

� The dentine-pulp complex

Dentine is a mineralised connective tissue that con-stitutes the main component of the tooth; it ensuressupport for the organ and confers elasticity. Seventyper cent of dentine is mineralised by hydroxyapatitecrystals; it is also composed of 20% organic matrixand 10% water. The organic component is mostlycomposed of proteins, which play various struc-tural, formative, signalling and homeostatic roles aswell as being important in the pulp healing process.Notably, dentine and bone are very similar in theircomposition but show some structural differences.

Dentine provides protection to the pulp, a softconnective tissue, which ensures the ‘vitality’ ofthe tooth1. The ‘dentine-pulp complex’ is so calledbecause of the intimate relationship of the two tis-sues that makes it difficult to separate their func-tional behaviours.

Dentine is a permeable tissue that is traversedby tubular structures called dentinal tubules,which extend from the enamel-dentine junction(EDJ) (or the cementum-dentine junction [CDJ] atthe root level) to the pulp cavity (chamber orcanal). These tubules contain dentinal fluid andthe odontoblast processes. The extent of theseodontoblast processes in the tubules remains con-troversial, with some authors claiming that theprocesses extend from the pulp chamber to theEDJ or CDJ2-4, whereas others suggest that theyare limited to the internal third of the tubules5-8.This structural distinction is important because thepresence of cellular extensions in the tubules caninfluence the therapeutic approach. Tubules thathave been partially occluded by cellular activitywill confer different permeability properties on thetissue than those whose dimensions have beenincreased by carious processes and this will affecttreatment planning accordingly.

The circumpulpal dentine is composed ofinter-tubular dentine (between tubules) and peri-or intra-tubular dentine (secondarily depositedwithin the tubule, which reduces the diameter ofthe tubule; Fig 1). The secretion of intra-tubulardentine is continuous throughout the life of thetooth and can be accelerated in certain patho-physiological conditions (such as cariousprocesses and tooth wear), leading to dentinalsclerosis.

The chemical composition of these two typesof dentine is different, particularly in terms of theircollagen contents and responses to etching proce-dures. These structural differences must be takeninto account clinically because, for example, theycan cause difficulties for bonding procedures. Pro-tocols must differ between bonding on scleroticdentine and filling a deep cavity in a relativelyyoung tooth. This structural difference is alsoimportant in endodontics with the introduction ofresin-based adhesive systems. Since the dentinestructure in the apical region is closer to a fibro-dentine structure than to a tubular one9, questionsremain regarding the sealing effectiveness of thesesystems in the short-, medium- and longer-termwhere the use of a protocol directly inspired bycoronal bonding may not be optimal for endodon-tic use.

ENDO (Lond Engl) 2009;3(3):171–184

172 � Simon et al Understanding pulp biology

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

� Three types of dentine

Confusion remains regarding the terminology ofthe different types of dentine, i.e. primary, second-ary and tertiary dentine. Many definitions havebeen proposed in the literature, several of whichare contradictory. Although these definitions havenot reached a consensus, they have been collatedby Goldberg and Smith10 who gave the followingdefinitions:

Primary dentine

Primary dentine is the earliest dentine formedduring tooth development, giving rise to thecrown and root structure of the tooth. It ‘patterns’the organ. The most external thin layer adjacent to the amelo-dentinal junction is formed as theodontoblasts are completing differentiation,which results in a variable tubular structure and isreferred to as mantle dentine.

Secondary dentine

Secondary dentine is physiologically secretedeither after the tooth has erupted into the oralcavity, or following apical closure. This dentine isgenerally responsible for the progressivelydecreasing space of the canal, which is improperly

called ‘calcification’. It is a physiological dentinerather than pathological in nature; its regularsecretion is responsible for the asymmetrical lossof endodontic volume. This biological processexplains the differences in canal volume betweena young and an older tooth. The secondary den-tine secretion is not uniform, but seems to occurpredominantly on the roof and lateral walls of thepulp chamber, and not on its floor. The chemicalcomposition and the histological structure of theprimary and secondary types of dentine are iden-tical. Only the rate of secretion differs: 4 μm/dayfor primary dentine and 0.4 μm/day for second-ary dentine. The interface between primary andsecondary dentine will sometimes be demarcatedby a subtle calciotraumatic line. However, sinceboth types of dentine are secreted by the sameodontoblasts there will be tubular continuity andthus, there does not appear to be any clinical con-sequence as to whether primary or secondary den-tine is present (Fig 2).

Tertiary dentine

Tertiary dentine is secreted in response to externalfactors, such as decay or abrasion, in order to pro-tect the underlying pulp. In the case of moderatestress, which does not cause any destruction ofodontoblasts, the secreted dentine is called ‘reac-

ENDO (Lond Engl) 2009;3(3):171–184

� 173Simon et al Understanding pulp biology

Fig 1 Schematic descriptionof the structure of circum-pulpal dentine, also calledorthodentine (Van Giesonstain, bar = 500 μm).

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

tionary dentine’; when the stress is more intenseand odontoblast survival is compromised (i.e. den-tine bridge formation at sites of exposure), it iscalled ‘reparative dentine’.

Understanding the distinction between thethree types of dentine is important, and above allthe distinction between secondary (physiological)and tertiary (healing) dentine must be clear,because they have generally been confused in theliterature.

� Dentine and bone

Many studies have shown that the compositionsof these tissues are more similar than previouslyappreciated. Some molecules that were consid-ered to be specific to dentine (such as dentinesialoprotein, DSP) have been found in alveolarbone. It is therefore more difficult to find specificmarkers to characterise these tissues. Neverthe-less, the profiles of these molecules show differ-ences between the two tissues.

On a structural level, bone and dentine havesome features in common. Cells of both bone anddentine have a similar mesodermal embryonicorigin, which may explain some of the similaritiesfound in their formation and structures. In bone,secretion of the matrix is performed by osteo-blasts; once embedded in the mineralised matrix,they appear to change into quiescent cells calledosteocytes which communicate with each otherthrough an extensive network of cell processes

embedded in canaliculi. In the dentine-pulp com-plex, odontoblasts remain on the formative sur-face of the dentine matrix but have a cell processextending through the dentine with many lateralprocesses. The similarities between the morpholo-gies of dentinal tubules and osteocyte canaliculihave recently been highlighted11 and recentmolecular data demonstrate striking similarities inthe behaviour of odontoblasts and bone cells atdifferent stages of their life cycles12.

� Pulpal cells

The pulp contains different types of cells, some ofwhich show specialisation or differentiation forparticular functions.

Odontoblasts

These are highly differentiated, post-mitotic cells,and are organised at the periphery of the pulp asa unicellular palisade. The presence of all the ele-ments of the secretory/mineralisation machineryin the cells confirms their intense activity, notablyduring primary dentinogenesis. At a later stageduring secondary dentinogenesis, the cells returnto a quiescent state, with a reduced number ofcytoplasmic organelles13. Cells are joined by cellu-lar junctions such as gap junctions, therebymaking a palisade of cells that provide a perme-ability barrier; gap junctions are also responsiblefor intra-cellular communication, which seems tobe involved in the pulp healing process14.

Unlike osteocytes, odontoblasts do not becomeincorporated in the matrix, except for their pro-cesses that are embedded in the tubule. This is whydentine must not be considered as an individualtissue but rather as the dentine-pulp complex. Theodontoblast processes contain limited organelles(which are supposedly responsible for the latersecretion of intra-tubular dentine), but are mostlyfilled by a dense network of microfilaments andother microtubules.

Increasingly, coronal odontoblasts are regardedas being different from those found in the root. Thecoronal odontoblasts are elongated and pyramidal,with an apical nucleus, whereas the radicular onesare more cubical, which is an indication of signifi-

ENDO (Lond Engl) 2009;3(3):171–184


Fig 2 The histologicalpattern of the twotypes of physiological(primary and second-ary) dentine.

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

cantly lower cellular activity. This fact is seldomtaken into account, but would explain why thera-peutic interventions that have been validated at acoronal level (pulp capping, for instance) will oftenfail when applied to the radicular pulp (pulpotomy).

The second layer of the pulp is a dense zone ofcells (the ‘Höhl’s layer’, separated from the odon-toblasts by an acellular layer called the ‘acellularWeil’s layer’). During tooth development, the cel-lular differentiation process requires a minimumnumber of mitotic divisions of the odontoblastprogenitor cells before they are competent to dif-ferentiate to the final cellular phenotype of theodontoblast. The last mitotic division has impor-tant spatial implications because it occurs perpen-dicular to the dental basement membrane result-ing in two daughter cells: the one next to thebasement membrane receives the inductive signalto differentiate to an odontoblast while the otherdoes not and contributes to the Höhl layer15. Thislayer has long been considered as a potentialreservoir of cells, containing incompletely differen-tiated cells that could be involved in the healingprocess for reparative dentinogenesis and dentinebridge formation if the odontoblast layer is dam-aged.

The capillary and nerve plexi that exist betweenboth layers are significant; only a few nerve fibresaccompany the cytoplastic extensions into thedentinal tubules and then only for a short distance.Capillaries are closely associated with the odonto-blast palisade and provide these cells with the nec-essary nutrients for their mineralising/syntheticactivities.

Pulp fibroblasts

Like all connective tissues, the major cell of pulptissue is the fibroblast; these cells are responsiblefor the formation and renewal of the extracellularmatrix, but at the same time they mediate its con-trolled remodelling. The extracellular matrix playsan important role in this connective tissue. Its vis-cosity changes with time (fibrosis increases withthe age of the tissue) and during pathophysiolog-ical processes. Its viscoelasticity enables it to adaptto potential (and moderate) variations in pressure,inherent in the inflammatory process. Thanks to

this adaptability, most episodes of pulp inflamma-tion are clinically silent. When the intra-pulpalpressure, connected with the vasodilatation inher-ent in inflammation, cannot be compensated any-more, the pain increases.

Immune cells

Dendritic cells and mast cells have been identifiedin the pulp tissue16, even in physiological condi-tions17. Macrophages are frequently found inhealthy pulp, especially in the periphery of thetissue18. These phagocytic cells participate in theimmune surveillance of the pulp, and enable arapid response to invading bacteria19,20. Productsof bacterial origin (such as toxins) diffuse via thetubules, and when in contact with pulp cells, theybehave like antigens; therefore, the immunesystem of the pulpal parenchyma plays an impor-tant role21. Dendritic cells capture antigens andmove these to the lymphatic nodes, where theyare presented to T lymphocytes. Then, these acti-vated T lymphocytes return to the damaged pulp.In this way, the host is immunised and will auto-matically respond to the future presence of theseantigens. Other molecules, such as those of thetransforming growth factor-beta (TGF-β) family,which have been liberated from the dentineduring the mineralisation process, are able to reg-ulate the immune system of the pulp22.

Dendritic cells also interact with nerve fibresand blood vessels within the pulp. The neuro-immunological response of the pulp is presumablythe first inflammatory reaction of the dentine-pulpcomplex17.

Dental pulp stem cells

The growing interest in stem cells by the scientificcommunity is also very apparent in dentalresearch. The discovery of dental pulp stem cells(DPSC)23 inside the pulp parenchyma demon-strated that the dental organ provides a ‘niche’ forreplacement cells. Another population of stemcells has also been discovered in the pulp of decid-uous teeth. These cells, or SHED (stem cells fromhuman exfoliated deciduous teeth)24, are particu-larly interesting because they are relatively easy to

ENDO (Lond Engl) 2009;3(3):171–184


Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

collect when the deciduous tooth is shed andreplaced by the permanent successor.

The presence of stem cells in the dental pulpoffers exciting possibilities for their exploitation inregenerative medicine both for dental and otherdiseases. They are easier to collect than bonemarrow cells, which was one of the main sourcesof post-natal stem cells. Also, they appear to be apromising reservoir of multipotential cells, and assuch offer significant potential for use in variousbiotechnological applications. The presence of apopulation of stem/progenitor cells in the dentalpulp provides a local source of cells for generatingnew ‘odontoblast-like cells’ for both natural pulpwound healing or regeneration and direct pulpcapping after injury to the tooth. It is still unclearwhether these stem cells have a developmentalderivation from the dental papilla or if theymigrate to the pulp through the vasculature. Animportant focus of future studies will be a moreprecise characterisation of these stem cells andtheir potential to allow their most effective appli-cation in new regenerative therapies.

The discovery of stem cells in the dental pulp hasbeen a significant advance for dentistry. Althoughmany questions remain unanswered, the identifica-tion of ‘post-natal stem cells’ in the tooth providesa new platform for the development of exciting bio-logical approaches for vital pulp therapy.

� Dentinal tubules – a means ofcommunication

� The tubular structure

The tubular density of dentine is high (30,000 mm2

on average), and these measure approximately 1to 3 μm in diameter on average in humans; theirdistribution is unequal throughout the dentine andtheir density increases near the pulp cavity, reflect-ing the crowding of odontoblasts as they movepulpally (Fig 3).

They course in a sinusoidal manner in thecrown rather than a rectilinear way, due to achange in the radius of curvature from the outerto the inner aspect of the dentine and havenumerous lateral extensions, which provide apotential means of communication betweenthem. Thus, much of the area of the dentinematrix is in direct communication with the odon-toblasts on its formative surface through theirprocesses. This has significant clinical implicationsin terms of the communication between the innerand outer regions of the tooth and how disease orsurgical intervention can have an impact on this.

The tubular density differs significantly at outerand inner areas of the dentine. It is estimated thatthe surface occupied by these tubules represents1% of the dentinal surface in the periphery of the

ENDO (Lond Engl) 2009;3(3):171–184


Fig 3 The density of thedentine tubules varies ac-cording to the depth of thedentine and the proximity tothe endodontic cavity.

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

dentine (under the enamel) and 22% near thepulp. This variable distribution underlines theimportance of location when drilling a cavity ormaking a coronal preparation, due to the openingof a diffusion pathway into the pulp tissue andpotentially allowing the ingress of bacteria, toxinsand other adverse agents.

The entire length of the tubules will containdentinal fluid, which probably represents a cellu-lar exudate from the pulp parenchyma togetherwith any local odontoblast secretions. Two differ-ent phenomena are encountered from themoment when open tubules are exposed.• Diffusion (Fig 4): When two biological environ-

ments (i.e. the pulp and the external environ-ment of the oral cavity) are separated by afilter (i.e. the tubular dentine), a concentrationgradient will be seen for agents in those twoenvironments. Diffusion occurs from the mostconcentrated to the least concentrated milieuin order to equilibrate the concentrations. Inthe case of teeth, the presence of bacteria athigh concentration in the saliva will lead totheir passive diffusion to the sterile pulpparenchyma. Nevertheless, the relatively largediameter of bacteria relative to the diametersof tubules is an obstacle to their movementthrough the tubules, although toxins and othermolecules can still readily pass through.

• The phenomenon of intra-pulpal pressure,which is greater than that external to the tooth:physiologically, the pressure in the pulp relativeto that outside the tooth is such that theretends to be an outward flow of fluid if opentubules are exposed, and thus limits the risk ofcontamination from inward diffusion (Fig 5).Fluid mechanics represents a complex science,especially in the context of the irregular archi-tecture within a dentinal tubule, but this out-ward pressure in the tubule ensures that thetubular dentine matrix does not act as a simplepermeable sieve.

The greater pressure intra-pulpally is directly rele-vant to clinical procedures in restorative dentistry.In the bonding process, it is recommended to ‘drybut not desiccate’ the dentinal surface. It is worthnoting that a few seconds after drying, the denti-nal surface is wet again. At this stage, it is not rec-ommended to further dry the surface otherwisedesiccation of the dentine may occur, causingunnecessary damage to the pulp, which wouldinevitably lead to post-surgical pain. New genera-tions of adhesives take this into account and tol-erate the presence of moisture in the dentine toensure optimal adhesion.

Dentinal permeability is an unavoidable factor totake into account in therapeutic procedures in den-

ENDO (Lond Engl) 2009;3(3):171–184


Fig 4 The principle of the diffusion phenomenon in thetubules.

Fig 5 The difference in the pressure between the intra-pul-pal cavity and the outside area is protective of the pulpparenchyma.

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

tistry. Surgical intervention with teeth will inevitablycause some damage to the pulp; the respondingdefence processes are numerous and complex andtheir interplay are not fully understood. Neverthe-less, they are the basis of the healing process, andpost-surgical pain is sometimes encountered aftereven conservative treatment. It is necessary torecognise the defence processes in order to opti-mally manage the post-treatment discomforts.

The pulpal hydrodynamic theory25 probablyprovides the best understanding of pulpal painand helps to explain how one tooth can becomevery sensitive to cold after a dentinal exposure,whereas other teeth in the same mouth remainpainless, or why for experimental purposes, appli-cation of a few drops of sugar solution can pro-voke violent pain whereas patients can toleratemuch more aggressive stimuli on other teeth? Onthe basis of histological and physiological observa-tions, Brännström demonstrated that dentinal sen-sitivity is closely associated with very fast and briefmovements of dentinal fluid inside the tubules,which may trigger responses within the vascularand neural structures at the pulpal level.

Since Brännström’s seminal publication, newtheories have been proposed to extend the under-standing of dentinal sensitivity, such as the possi-bility of partial innervation of the tubules. Recentfindings suggest the possible existence of sensoryproperties for the odontoblast and its role in sen-sory transmission of stimuli to the tooth is a topicof much study26,27.

Although the precise mechanisms of pain trans-mission in teeth remain to be fully elucidated, pres-ent knowledge already allows understanding ofsome of the clinical responses encountered in day-to-day practice. It is quite common to find patientscomplaining of pain after they had received abonded inlay using an indirect technique. Sensitiv-ity is mainly encountered while chewing and can beexplained by the hydrodynamic theory. Whenchewing, some bondings have a ‘shock absorber’effect owing to their viscoelastic properties. Duringeach micro-movement, fluid movements in thetubules and in the hybrid layer are triggered andmay be responsible for the extra external pressurebeing exerted on the pulp (Fig 6). Thus, acute paincan be the result of routine restorative treatment.

Movement of the dentinal fluid may explain thepeculiar pain described by patients presenting witha root crack or fracture. This pain is considered aspeculiar as it is not a reaction to pressure, but it isfelt during relaxation (opening). In the case of acleft, the pressure within the tooth during occlusioncauses micro-movements and tends to separate thetwo edges of the cleft; therefore, the pulpal inter-stitial fluid tends to invade the space created. Whenpressure is released, the two dentinal edges willclose up, thus creating increased intra-pulpal pres-sure resulting in very sharp and transient pain.Therefore, the appropriate treatment for suchpathology is to retain the edge gap and provide atooth covering (for example, with a crown). In thiscase, endodontic treatment has little merit.

ENDO (Lond Engl) 2009;3(3):171–184


Fig 6 The principle of Brännström’s hydrodynamic theory: (a) the application of forces on the filling can lead to a displacement of the dentinal fluid intothe tubuli, and create an over-pressure in the pulp, which can cause post-surgical discomfort (b and c).

a b c

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

� Consequences of new technological developments

Knowledge of the dentine-pulp complex providesan important foundation for a new era of dentistrywith a biological focus. For example, the conceptof cavity disinfection is of primary importance toprevent bacterial penetration and diffusion oftoxins beneath restorations and this is beingexploited in the development of new restorativeproducts. Bonding systems have now been devel-oped, which aim to complement the adhesiveproperties with antibacterial molecules to controlresidual and recurrent caries. Clearfil™ ProtectBond (Kuraray, Frankfurt, Germany), in which 12-methacryloyloxy-dodecylpyridinium bromide(MDPB) has been added to the bonding agent, isa relevant example of this new generation of prod-ucts. New research is underway to consider theaddition of other molecules, which would play arole in stimulating pulpal healing.

� Towards a biological approach

The concept of vital pulp therapy is one thatfocuses on using biological principles to maintainpulp vitality. This requires an understanding of themany pathophysiological processes of the den-tine-pulp complex and the development of newmaterials for clinical use, which exploit the pulpalhealing processes and better mimic the physiolog-ical tissues being restored. However, this evolutionof new therapeutic principles within conservativedentistry dictates that techniques should be devel-oped in which new materials are used in the con-text of minimising damage to the dental tissuesduring restorative preparation.

The gradual replacement of amalgams bycomposites, which are considered to have betteraesthetic properties in coronal restorations hasbeen justified in part by the toxicity of the formerboth to the patient and environment, althoughresins are far from being inert themselves. How-ever, adhesive restorations have a biologicaladvantage in minimising the amount of dentaltissue needing to be removed during preparation.Apart from the biomechanical advantages of max-

imal retention of dental tissue, injury to the tissuesis minimised and diffusion pathways to the pulpare reduced with the decreased number of opentubules. The improvement in the long-term prog-nosis offered by these approaches is evident28.Therefore, minimalist dentistry, also called ‘non-invasive’ dentistry or Minimal Intervention Ther-apy, has significant potential8,29.

� Pulpal responses to injury

Progress in tissue regeneration has enabledresearchers to understand better how the odonto-blasts and, more generally, the pulp react after aninjury. The tertiary dentine secreted in the absenceof pulpal exposure is commonly reactionary innature and helps to both restore the structuralintegrity of the tooth and increase the distancebetween the injurious agent and the pulp. After theinitial secretion and development of the primarydentine, the secretory odontoblast seems to fall intoa semi-quiescent, semi-inactive state during whichit continues its secretory activity but at a muchslower rate. This process of secondary dentinogen-esis is responsible for the gradual reduction in sizeof the pulp chamber, although the molecular con-trol of the odontoblasts responsible for the decreasein activity remains to be elucidated.

During injury to the tooth (caries, trauma orwear), a cascade of pulpal responses will be initi-ated. The exquisite regenerative or healing poten-tial of the pulp though means that transient histo-logical changes do not necessarily lead to clinicallysignificant manifestations30. Depending on thenature of the injury (whether it is brief or pro-longed, or low or high intensity), the scope of thepulpal responses will likely differ. Injury of weak ormoderate intensity will often be resolved by a briefinflammatory response followed by reactionarydentinogenesis. With injury of a greater intensity,odontoblast death may well ensue (e.g. deepcaries or severe trauma), and as long as inflamma-tion does not become uncontrolled, differentiationof a new generation of odontoblast-like cells mayoccur leading to dentine bridge formation at sitesof exposure: this process is called reparativedentinogenesis (Fig 7).

ENDO (Lond Engl) 2009;3(3):171–184


Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

� Reactionary dentinogenesis

After injury, odontoblasts abandon their quiescentstate and begin to secrete again at a faster pacedepositing reactionary dentine. Histologically, thecalciotraumatic line signals the beginning of thisnew activity in the dentine matrix (Fig 8). Impor-tantly, because the same cells are responsible forsecretion of the reactionary dentine, tubular con-tinuity is seen together with consequent mainte-nance of dentine permeability. This clearly hasconsequences in regard to the choice of restora-tive material, which should not allow leaching ofany cytotoxic components.

Although still poorly understood, it can be pos-tulated that the molecular control processes thatare responsible for switching off the odontoblastduring the change from primary to secondarydentinogenesis may be reversed to stimulate theodontoblasts again. Decryption of the phenome-non of cell reactivation is necessary, and will allowdevelopment of new therapeutics that control cellactivity.

It is also important to consider the nature of thesignalling process between the injurious agent andthe odontoblasts. Bacteria and their toxins are keycandidates in the direct stimulation of odonto-blasts during caries31. Also, lipopolysaccharidesand other bacterial toxins initiate intra-pulpalinflammatory processes, but other signalling

processes may also be critical in the overall balanceof pulpal cell responses leading to healing andtissue regeneration32,33.

Dentine is a mineralised connective tissue rich incollagen, but it also contains trace amounts of verypotent bio-active molecules including cytokines andgrowth factors, which are sequestered within thematrix during the mineralisation process andbecome essentially fossilised therein. During thedecay process, demineralisation of the tissue isaccompanied by the release of these molecules,which were initially fossilised34. In this pool of sub-stances, many growth factors can be found, espe-cially those of the TGF-β family35,36. These growthfactors have a variety of cell signalling propertiesand act at very low concentrations. Once liberated,these factors traverse the tubules to the pulp andinduce various cellular responses, including activa-tion of the odontoblasts37. Once stimulated, theseformerly quiescent cells enter an active state, andsecrete tertiary reactionary dentine (Fig 9).

From this concept, it is possible to imagineopportunities for therapeutic stimulation, inducinga targeted release of these proteins. For example,cleaning the cavity with EDTA solution, which iswell known for its ability to dissolve the mineralphase, would be a potential way to liberategrowth factors and to induce the stimulation ofodontoblasts38-40. Etching with orthophosphoricacid, used for conditioning the dentine in bonding

ENDO (Lond Engl) 2009;3(3):171–184


Fig 7 The two kinds of tertiary dentinogenesis. Fig 8 Frontal histological section of a mouse molar whichhas been treated with a coronary filling. The staining of re-actionary dentinogenesis is more pronounced than the otherdentine.

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

procedures, also promotes demineralisation of thedentine and liberation of biological factors. Otherproducts that are presently in the dentist’s thera-peutic arsenal might come back into favour withnew indications. For a long time, calcium hydrox-ide had been used as a protective lining, especiallybeneath amalgam fillings, but has more recentlyfallen out of favour. Nevertheless, this materialcertainly has the ability to release componentsfrom the dentine, including growth factors41.Unlike the chelating agents which only have briefcontact with the dentine, calcium hydroxideremains in place beneath restorations and favoursa gentle and continued dissolution, thus releasinggrowth factors; its action is prolonged and poten-tially controllable depending on the form of theproduct.

More recently, the action of mineral trioxideaggregate (ProRoot™ MTA, Dentsply Maillefer,Ballaigues, Switzerland) in releasing growth fac-tors has been demonstrated42, although theamounts released differ from those liberated bycalcium hydroxide. These differences are interest-ing because they might explain the differences inthe behaviour of both materials. If the processesunderlying this action were better understood,then the use of such linings under coronal restora-tions might readily find favour again, soon therebyproviding a new focus for research in bioactivematerials.

� Reparative dentinogenesis

Odontoblasts are the only cells that secrete den-tine. If they suffer injury, the formation of a den-tine bridge at sites of pulpal exposure is still possi-ble, providing that new odontoblast-like cells are‘available’ in the area. Conventionally, woundhealing involves cell migration from the edge ofthe injury and with cell division, the superficialcells move to the centre of the injury regeneratingand repairing the tissue and allowing its reorgani-sation.

Odontoblasts are differentiated post-mitoticcells, and are unable to divide to produce newsecretory cells. When these cells are lost, anotherform of replacement occurs involving stem or pro-genitor cells resident in the pulp43,44 (Fig 10). Afterpulp exposure, and after placement of an appro-priate material, a dentine bridge is formed in a fewweeks (Fig 11) by ‘new odontoblast-like cells’.The origin of these odontoblast-like cells in thedentine pulp healing process is not clearly estab-lished. Several authors believe that these processesare likely to be the same as those involved in toothdevelopment45; however, the origin of these cellsis still unclear and an origin from outside the toothvia the vasculature cannot be excluded.

Various clinical procedures for repair of a pulpexposure have been proposed and calciumhydroxide has long been considered as the mate-

ENDO (Lond Engl) 2009;3(3):171–184


a b c

Fig 9 Numerous matrix proteins are fossilised in the collagen matrix of the dentine during the mineralisation process: (a) these factors are released bythe dissolution of the mineral matrix (whether pathological or therapeutic); (b) they join the odontoblast layer via the tubules; and (c) nowadays, theyare considered to be a potential signalling pathway in the dentine pulp healing process.

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

rial of choice. However, the poor quality of thedentinal bridge and its lack of a hermetic seal arepotential sources of failure with this material.There is still a lack of trust in pulp capping due tothe inability to predict prognosis and practitionerswill frequently remove the pulp of the tooth ratherthan try to keep it alive. Several in vivo studieshave shown that MTA induces the creation of adentine bridge of good quality, with an effectivehermetic seal, which can merge with the dentinalwalls at the edge of the defect46. The advantage ofthe quality of the dentine bridge obtained withMTA versus calcium hydroxide as a pulp cappingagent has been recently demonstrated in a ran-domised clinical trial47. Although several authorshave proposed capping the pulp with the fillingmaterial itself, there is no histological evidence that

this can induce dentine bridge formation. Even if,clinically, the results seem to be satisfactory, thisapproach cannot be considered to be reliable in thelong term. This example perhaps illustrates whatdistinguishes the clinician from the scientist.Whereas clinicians focus on the sealing propertiesof materials and the prevention of any bacterial orfluid leakage, scientists are more interested in thebio-activity of these materials and their ability toinduce a wound healing response. Both are com-plementary, and it is essential that these twoapproaches are brought together. For the future,existing materials will provide an important step-ping stone towards the development of more spe-cific biomolecules as dentistry develops in abiotechnological direction and may encouragegreater practitioner uptake of such approaches.

ENDO (Lond Engl) 2009;3(3):171–184


Fig 10 Reparative dentinogenesis. Contrary to conventionalconnective tissues, the healing process in the odontoblasticlayers does not occur by cell division at the edge of the injury(a and b). The attraction of new cells and their differentiationinto dentine secreting cells (c) induce the formation of adentinal bridge in contact with an appropriate material (e).

a b

c d

e

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

� The clinical consequences

Translation of biological knowledge into the clin-ical setting can simply involve the adoption of amore biological frame of mind during treatmentplanning, but it is not always that easy. Forinstance, the impact of tooth structure on biolog-ical events can be fairly readily appreciated, suchas in the thickness of the residual dentine sepa-rating a cavity from the pulp which can be animportant factor in its protection. In deeper cavi-ties, where the thickness of the cavity floor is lessthan 0.5 mm, the number and the height of the‘opened’ tubules is such that the communicationwith the pulp is possibly comparable to that of anactual cavity exposure48. In contrast, clinically, itcan be very difficult, if not impossible, to knowthe inflammatory status of the pulp, and thecytological state of the odontoblasts since pulpalhistology does not necessarily correlate well withclinical presentation. This makes it very difficultto effectively plan treatment in such situationsand there is an urgent need for more reliablemeans of diagnosing of the activity of cariouslesions and the level of pulpal inflammation.While thermal or electrical tests provide informa-tion on the innervation of the pulp, there is aneed for tests that evaluate the vasculature and,in particular, the effects thereon of any inflam-mation that may be present. Unfortunately, thehard tissue shell enclosing the pulp constrainsthe use of techniques such as Laser Dopplerscanning or scintigraphy, which provide valuableinformation in other medical specialities.

� Conclusions

The application of biotechnology to the field ofrestorative dentistry has immense potential. Formany years, there has been a gap between basicresearch and dental practice, but the recent arrivalof new adhesive systems containing antibacterialmolecules provide a first step towards biologicaltreatment of the pulp. It is important that researchin dentistry goes beyond the study of alloys, sizesof drills, disinfecting power of solutions, ormechanical resistance and embraces the opportu-

nities offered by biotechnology, which will changethe perception and practice of dentistry.

Above all, the training of dentists will have toadapt to these new approaches to dental treat-ment. Progress has been made in the last tenyears in aesthetic management and restoration offunctionality. Very soon, the modern dentist willhave to be aware that biological approaches,rather than the purely mechanical functions, willadd a new dimension to restorative dentistry.

� References1. Linde A, Goldberg M. Dentinogenesis. Crit Rev Oral Biol

Med 1993;4:679-728.2. Maniatopoulos C, Smith DC. A scanning electron micro-

scopic study of the odontoblast process in human coronaldentine. Arch Oral Biol 1983;28:701-710.

3. Sigal MJ, Aubin JE, Ten Cate AR, Pitaru S. The odontoblastprocess extends to the dentinoenamel junction: an im-munocytochemical study of rat dentine. J HistochemCytochem 1984;32:872-877.

4. Sigal MJ, Aubin JE, Ten Cate AR. An immunocytochemicalstudy of the human odontoblast process using antibodiesagainst tubulin, actin, and vimentin. J Dent Res 1985;64:1348-1355.

5. Thomas HF, Carella P. Correlation of scanning and transmis-sion electron microscopy of human dentinal tubules. ArchOral Biol 1984;29:641-646.

6. Thomas HF, Carella P. A scanning electron microscope studyof dentinal tubules from un-erupted human teeth. ArchOral Biol 1983;28:1125-1130.

7. Garberoglio R, Brannstrom M. Scanning electron micro-scopic investigation of human dentinal tubules. Arch OralBiol 1976;21:355-362.

8. Pashley DH. Pulpodentin Complex. In: Hargreaves KM,Goodis HE (eds). Seltzer and Bender’s Dental Pulp. Chicago:Quintessence Publishing, 2002:63-93.

ENDO (Lond Engl) 2009;3(3):171–184


Fig 11 Dentine bridge 5 weeks after pulp capping with MTA of a mouse first molar(frontal semi-thin section, x50 magnification, methylene blue-azur II).

Copyrig

ht

by

N

otfor

Qu

in

tessence

Not

forPublication

9. Mjör IA, Sveen OB, Heyeraas KJ. Pulp-dentin biology inrestorative dentistry. Part 1: normal structure and physiolo-gy. Quintessence Int 2001;32:427-446.

10. Goldberg M, Smith AJ. Cells and extracellular matrices ofdentin and pulp: a biological basis for repair and tissue en-gineering. Crit Rev Oral Biol Med 2004;15:13-27.

11. Lu Y, Xie Y, Zhang S, Dusevich V, Bonewald LF, Feng JQ.DMP1-targeted Cre expression in odontoblasts and osteo-cytes. J Dent Res 2007;86:320-325.

12. Simon SR, Smith AJ, Lumley PJ, Berdal A, Smith G, Finney S,Cooper PR. Molecular characterisation of young and matureodontoblasts. Bone 2009 (in press).

13. Jones S, Boyde A. In: Linde A (ed). Dentin and Dentin-ogenesis. Boca Raton: CRC Press, 1984:81-134.

14. Magloire H, Couble ML, Romeas A, Bleicher F. Odontoblastprimary cilia: facts and hypotheses. Cell Biol Int 2004;28:93-99.

15. Ruch JV, Lesot H, Begue-Kirn C. Odontoblast differentia-tion. Int J Dev Biol 1995;39:51-68.

16. Jontell M, Gunraj MN, Bergenholtz G. Immunocompetent cellsin the normal dental pulp. J Dent Res 1987;66:1149-1153.

17. Jontell M, Okiji T, Dahlgren U, Bergenholtz G. Immune de-fence mechanisms of the dental pulp. Crit Rev Oral BiolMed 1998;9:179-200.

18. Trowbridge HO. Histology of Pulpal Inflammation. In:Hargreaves KM, Goodis HE (eds). Seltzer and Bender’s DentalPulp. Chicago: Quintessence Publishing, 2002:227-245.

19. Okiji T, Kawashima N, Kosaka T, Matsumoto A, KobayashiC, Suda H. An immunohistochemical study of the distribu-tion of immunocompetent cells, especially macrophagesand Ia antigen-expressing cells of heterogeneous popu-lations, in normal rat molar pulp. J Dent Res 1992;71:1196-1202.

20. Okiji T, Morita I, Suda H, Murota S. Pathophysiologicalroles of arachidonic acid metabolites in rat dental pulp. ProcFinn Dent Soc 1992;88(Suppl 1):433-438.

21. Heyeraas KJ, Sveen OB, Mjor IA. Pulp-dentin biology inrestorative dentistry. Part 3: Pulpal inflammation and its sequelae. Quintessence Int 2001;32:611-625.

22. Jontell M, Bergenholtz G, Scheynius A, Ambrose W. Dendriticcells and macrophages expressing class II antigens in the normal rat incisor pulp. J Dent Res 1988;67:1263-1266.

23. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S.Postnatal human dental pulp stem cells (DPSCs) in vitro andin vivo. Proc Natl Acad Sci U S A 2000;97:13625-13630.

24. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG,Shi S. SHED: stem cells from human exfoliated deciduousteeth. Proc Natl Acad Sci U S A 2003;100:5807-5812.

25. Brännström M. A hydrodynamic mechanism in the trans-mission of pain producing stimuli through the dentin. In:Anderson DJ (ed). Sensory mechanisms in dentine. London:Pergamon Press, 1963:73-79.

26. Allard B, Magloire H, Couble ML, Maurin JC, Bleicher F.Voltage-gated sodium channels confer excitability to hu-man odontoblasts: possible role in tooth pain transmission.J Biol Chem 2006;281:29002-29010.

27. Okumura R, Shima K, Muramatsu T, Nakagawa K, ShimonoM, Suzuki T, et al. The odontoblast as a sensory receptorcell? The expression of TRPV1 (VR-1) channels. Arch HistolCytol 2005;68:251-257.

28. Murray PE, About I, Lumley PJ, Smith G, Franquin JC, SmithAJ. Postoperative pulpal and repair responses. J Am DentAssoc 2000;131:321-329.

29. Pashley DH. Dynamics of the pulpo-dentin complex. CritRev Oral Biol Med 1996;7:104-133.

30. Seltzer S, Bender IB, Ziontz M. The dynamics of pulp in-flammation: correlations between diagnostic data and actu-al histologic findings in the pulp. Oral Surg Oral Med OralPathol 1963;16:969-977.

31. Durand SH, Flacher V, Romeas A, Carrouel F, Colomb E, Vin-cent C, et al. Lipoteichoic acid increases TLR and functionalchemokine expression while reducing dentin formation in invitro differentiated human odontoblasts. J Immunol2006;176:2880-2887.

32. Magloire H, Bouvier M, Joffre A. Odontoblast responseunder carious lesions. Proc Finn Dent Soc 1992;88(Suppl1):257-274.

33. Tziafas D, Smith AJ, Lesot H. Designing new treatmentstrategies in vital pulp therapy. J Dent 2000;28:77-92.

34. Smith AJ. Vitality of the dentin-pulp complex in health anddisease: growth factors as key mediators. J Dent Educ2003;67:678-689.

35. Cassidy N, Fahey M, Prime SS, Smith AJ. Comparative analy-sis of transforming growth factor-beta isoforms 1-3 inhuman and rabbit dentine matrices. Arch Oral Biol1997;42:219-223.

36. Smith AJ, Matthews JB, Hall RC. Transforming growthfactor-beta1 (TGF-beta1) in dentine matrix. Ligand activa-tion and receptor expression. Eur J Oral Sci 1998;106(Suppl1):179-184.

37. Smith AJ, Tobias RS, Cassidy N, Begue-Kirn C, Ruch JV,Lesot H. Influence of substrate nature and immobilizationof implanted dentin matrix components during induction ofreparative dentinogenesis. Connect Tissue Res 1995;32:291-296.

38. Smith AJ, Tobias RS, Cassidy N, Plant CG, Browne RM,Begue-Kirn C, et al. Odontoblast stimulation in ferrets bydentine matrix components. Arch Oral Biol 1994;39:13-22.

39. Smith AJ, Tobias RS, Murray PE. Transdentinal stimulation ofreactionary dentinogenesis in ferrets by dentine matrix com-ponents. J Dent 2001;29:341-346.

40. Smith AJ, Tobias RS, Plant CG, Browne RM, Lesot H, RuchJV. In vivo morphogenetic activity of dentine matrix proteins.J Biol Buccale 1990;18:123-129.

41. Graham L, Cooper PR, Cassidy N, Nor JE, Sloan AJ, Smith AJ.The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials 2006;27:2865-2873.

42. Tomson PL, Grover LM, Lumley PJ, Sloan AJ, Smith AJ, CooperPR. Dissolution of bio-active dentine matrix components bymineral trioxide aggregate. J Dent 2007;35:636-642.

43. Fitzgerald M. Cellular mechanisms of dentinal bridge repairusing 3H-thylidine. J Dent Res 1979;58:2198-2206.

44. Fitzgerald M, Chiego DJ Jr, Heys DR. Autoradiographicanalysis of odontoblast replacement following pulp exposurein primate teeth. Arch Oral Biol 1990;35:707-715.

45. Mitsiadis TA, Rahiotis C. Parallels between tooth develop-ment and repair: conserved molecular mechanisms followingcarious and dental injury. J Dent Res 2004;83:896-902.

46. Simon S, Cooper P, Smith A, Picard B, Ifi CN, Berdal A. Eval-uation of a new laboratory model for pulp healing: prelimi-nary study. Int Endod J 2008;41:781-790.

47. Nair PN, Duncan HF, Pitt Ford TR, Luder HU. Histological,ultrastructural and quantitative investigations on theresponse of healthy human pulps to experimental cappingwith mineral trioxide aggregate: a randomized controlledtrial. Int Endod J 2008;41:128-150.

48. Murray PE, About I, Lumley PJ, Franquin JC, Remusat M,Smith AJ. Cavity remaining dentin thickness and pulpal activ-ity. Am J Dent 2002;15:41-46.

ENDO (Lond Engl) 2009;3(3):171–184


Understanding pulp biology for routine clinical practiceResults: Novel biotechnological insights...

Documents

Transcript of Understanding pulp biology for routine clinical practiceResults: Novel biotechnological insights...