mary teeth is still lacking. Histological methodshave not clearly revealed the nature of themicro-environment of the sites of initiation ofthese forms of pulpal calcification since theearliest sites are not resolved in the lightmicroscope. It has been suggested that deador inflamed cells, bacteria, matrix fibres orblood vessels and nerves form foci for calcifi-cation [2-5]. Limited information is availableregarding the prevalence and long-term prog-nosis of pulp calcification in injured primaryteeth [5,6]. Neither are there any studies avail-able of the chemical abundance of Ca and P inreparative dentin .

The purpose of this study was to describe thedetailed structure and chemical composition ofreparative dentin in primary teeth using lightmicroscopy (LM), scanning electron microscopy(SEM and X-ray microanalysis (XRMA).

M AT E R I A L A N D M E T H O D SStudy Group 1The material comprised 22 traumatized pri-mary teeth extracted due to trauma immedi-ately or due to complications after traumaticdental injuries. The acute diagnoses includedsubluxation, extrusive luxation and lateral lux-ation. Inclusion criteria were: 1. 5/6 full rootlength; and 2. Presence of reparative dentin.

B I O G R A P H YAgneta Roberston is anassociate professor andspecialist in pediatricdentistry at the Instituteof Odontology in Göte-borg, Sweden. She is thehead of the departmentof pediatric dentistry and the specialist clinicin pediatric dentistry. Her main interest inresearch concerns clinical and histologicalstudies of traumatized primary and youngpermanent teeth.

A B S T R A C TThe purpose of this study is to describe thedetailed structure and chemical compositionof reparative dentin in primary teeth usinglight microscopy, scanning electronmicroscopy and X-ray microanalysis. Thestudy comprised decalcified sections of 22traumatized and undecalcified sections of 8primary teeth. The teeth displayed normalpulp tissue morphology, but reduced in sizedue to the reparative dentin. Five differentconfigurations of reparative dentin wereidentified on the walls of the pulp cavity.Lower values in reparative dentin comparedwith normal dentin were found for Ca, Pand the ratio Ca/C.

K E Y W O R D Slight microscopy, histology, scanning elec-tron microscopy, X-ray microanalysis, energydispersive X-ray spectroscopy, Brown andBrenn staining, demineralized sections,dentin, reparative dentin, primary teeth-

A U T H O R D E TA I L SDr Agneta Robertson, Department of Pedodontics, Institute of Odontology at the SahlgrenskaAcademy,University of Gothenburg, Box 450, SE-405 30 Göteborg, SwedenTel: +46 31 786 3144e-mail: agneta.robertson@vgregion.se

Microscopy and Analysis 23(4):13-16 (EU),2009

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I N T R O D U C T I O NAll primary and permanent teeth show a layerof secondary dentin around the pulp cavity,which is distinct from the regular primarydentin. It is often rather difficult to distinguishbetween primary and secondary dentin [1].Secondary dentin comprises the circumpulpalportion of regular dentin and the primary oneproduced circumpulpally throughout the laterperiods of the vital tooth [1]. However, repar-ative dentin differs in many respect from pri-mary and secondary dentin and is thus easilydistinguished from these [1].

The formation of reparative dentin is animportant protective function of the dentalpulp in response to dentin injury. Calcificationof the pulp is common at all ages, but cariesand traumatic injuries affecting the pulp areknown to increase their incidence [2-4]. Trau-matic injuries often give rise to an accelerationof hard tissue formation within the pulp cavity,sometimes leading to complete calcification[7]. Frequent efforts have also been made tostudy the incidence of reparative dentin as afunction of various types of injury [2-4]. Pulpalcalcifications have been studied by clinical,radiographical, histological and scanning elec-tron microscopical techniques but informationabout the nature of pulp calcification in pri-

Histomorphology and X-Ray Microanalysisof Reparative Dentin in Primary TeethA. Robertson,1 and S. Nietzsche2 1. Department of Pedodontics, Institute of Odontology, University of Gothenburg, Göteborg, Sweden. 2. Centre of Electron Microscopy, Friedrich-Schiller-Universität Jena, Germany

Figure 1: Photomicrograph of an undemineralizedsection of a primary incisor with reparativedentin. The locations for the XRMA mea-surements in normal and reparative dentin,respectively, are marked with squares. Scale bar =100 µm.

MICROSCOPY AND ANALYSIS MAY 2009 13

Study Group 2The material comprised eight exfoliated pri-mary teeth. The inclusion criterion was thepresence of reparative dentin.

Light Microscopy The teeth were fixed and stained immediatelyafter extraction in 10% neutral-buffered for-malin. All teeth were decalcified with EDTA,conventionally prepared for paraffin embed-ding and then serially sectioned and stainedwith hematoxylin and eosin (H & E).

The teeth were evaluated by lightmicroscopy and were analyzed according topredetermined parameters:Odontoblast layer: 1. Normal structure,reduced or missing. 2. Regular, irregular layer.Pulp tissue morphology: Normal, altered tissue Primary and secondary dentin: Regular, irreg-ular dentinReparative dentin: 1. Localization. 2. Regular,irregular dentin. 3. Denticles. 4. Diffuse calcifi-cations

Scanning Electron Microscopy and X-RayMicroanalysisAll twenty sections from the primary teethpreviously examined by LM were analyzed inSEM. The slides were kept in xylene till thecover glass could easily be removed. Aftermounting on sample holders for the micro-scope the sections were sputter coated withgold. They were then examined in a Zeiss (LEO)Gemini IMB 1530 field-emission scanning elec-tron microscope at 5 kV and 7 kV.

Six undemineralized primary incisors weresectioned sagittaly in a bucco-lingual directionwith a Leitz low speed saw microtome. Afterexamination in an Olympus polarizing micro-scope employing strain-free objectives, thesections were mounted on sample holders forthe SEM and sputter coated with carbon. Forthe XRM analysis a Philips SEM 515 with anEDAX DX4 ECON detector was used. Measure-ments of C, O, P and Ca were performed in twolocations in reparative and normal dentin,respectively (Figure 1). For all measurementsthe X-rays were detected by a small window(6.134.3 µm) at a magnification of 6553. Therelative amounts of C, O, P, and Ca were calcu-lated with the Point Electronic DISS 2 program.All values are to be regarded as semi-quanti-tative.

In the SEM analysis the following criteriawere evaluated: Primary and secondarydentin: Regular, irregular dentin. Interfacebetween the dentin (primary and secondary)and the reparative dentin: Regular, irregulardentin. Reparative dentin: 1. Localization. 2.Regular, irregular dentin

Statistical AnalysisThe data from XRMA were compiled in anExcel spreadsheet and the Mann-Whitney sta-tistical test was used to compare median val-ues for C, O, P, Ca and the ratios Ca/P and Ca/C.

R E S U LT SLight MicroscopyOdontoblast layer: The odontoblast layercould be observed in all teeth. The amount of

layers varied from one single layer to three orfour. The layers were often irregular (Figure2).Pulp tissue morphology: The teeth displayednormal pulp tissue morphology, but it wasreduced in size due to the reparative dentin.The number of cells, predominately odonto-blasts, in the parts of the pulps was neverthe-less decreased in the most cases.Primary and secondary dentin: The primaryand secondary dentin look regular and therewas no marked border between the layers(Figure 3).Reparative dentin: Five different configura-tions of reparative dentin were identified onthe walls of the pulp cavity (Figure 4): 1. Hardtissue in the most coronal part of the pulp

chamber. 2. Hard tissue in the most coronalpart of the pulp chamber in addition to hardtissue along one of the lateral pulp canal walls.3. Hard tissue in the most coronal part of thepulp chamber in addition to both the lateralpulp canal walls. 4. An isolated formation ofnew hard tissue on the lateral pulp canal wall.5. Hard tissue filling up a substantial portion ofthe coronal pulp chamber.

The reparative dentin had formed in thepulp to a varying extent. The most frequentlyoccurring types of hard tissue formation weretype 2 and 4. Interglobular dentin wasobserved and in some teeth incremental lineswith alternating high and low mineral contentwere seen.Denticles: In a few teeth free denticles were

Figure 2: Photomicrograph of the odon-toblast layers. Scale bar=100 µm.

MICROSCOPY AND ANALYSIS MAY 200914

Figure 3: Photomicrograph (a) and SEMimage (b) of regular primary andsecondary dentin with nomarked border between thelayers.

NB: Move scale barwith Photoshop

NB: Move scale baron SEM image withPhotoshop

a

b

DE N T I N I N PR I M A R Y TE E T H

observed (Figure 5). The sizes of the denticlesin the pulp of the teeth were observed to varygreatly. The size of these was sometimes sosmall as to be barely perceptible, while othersconsisted of large conglomerate fused masses.Those denticles were partly lined with anodontoblast like cell layer. The calcificationappeared to consist of discrete smooth-sur-faced laminated denticles or irregularlyshaped non-laminated denticles. Denticleswithout laminations often appeared with anirregular outline compared with the lami-nated denticles. The denticles that consistedof distinct concentrically arranged lamellas didnot contain dental tubules. The denticles con-tained blood and nerves.Diffuse calcifications: All teeth show diffusecalcifications which were distinct and occurredas unorganized masses throughout the pulp(Figure 2). They were often related to bloodvessels or nerves.

Scanning Electron MicroscopyThe primary and secondary dentin: The dentinformed was regular and well mineralized. Thedentin exhibits tubules with a diameter of 3µm or less (Figure 3b).The interface between the primary and sec-ondary dentin and the reparative dentin: Theinterface between the secondary dentin andreparative dentin was rough and diffuse. Thedentin tubules changed direction passing theinterface (Figure 3b).The reparative dentin: The dentin formedlooked with few exceptions irregular andthere were a reduced number of tubules. In afew cases the tubules there were sparse andappeared regular and twisted. The number oftubules was reduced on the side of the walls ofthe pulp while there was no marked reductionin others. In the reparative dentin formed inthe horn of the pulp there was no markedreduction in the number of tubules, in somecases. Some tubules could be followed fromthe primary and secondary dentin into thereparative dentin and a change in the direc-tion of the tubules was often noted. Thetubules were either empty or filled with odon-toblast processes. They had a variable size anddistribution; in addition, occluded tubuleswith a high mineral content were seen. Insome cases the reparative dentin looked morebone-like than dentin-like and there were alsointermediate forms. Denticles: The denticles had the appearance ofosteodentin with cell inclusions in a ring-likeformation. The bone-like tissue had lacunae-like cavities, free from cells, scattered through-out the tissue.

X-Ray MicroanalysisAll median and mean values are given in Table2. There were no differences between C and Oin normal and reparative dentin, respectively.The Ca and P values were significantly lower(p<0.05) in reparative dentin compared withnormal dentin, however, the ratio Ca/P did notdiffer between normal and reparative dentin.The ratio Ca/C was significantly lower (p<0.01)in reparative dentin compared with normaldentin.

MICROSCOPY AND ANALYSIS MAY 2009 15

Figure 4: Photomicrographs of five different configurations of reparative dentinidentified on the walls of the pulp cavity. Scale bars: (a) = 50 µm; (b-e) =100 µm.(a) Hard tissue in the most coronal part of the pulp chamber (b) Hard tis-sue in the most coronal part of the pulp chamber in addition to hard tis-sue along one of the lateral pulp canal walls.(c) Hard tissue in the most coronal part of the pulp chamber in addition toin the lateral pulp canal walls.(d) An isolated formation of new hard tissue on the lateral pulp canal wall. (e) Hard tissue filling a substantial portion of the coronal pulp chamber.

D I S C U S S I O NThis study has shown that the reparativedentin formation, described by comparinglight microscopy and scanning electronicmicroscopy, was irregular and with a reducednumber of tubules. The chemical analysesrevealed lower values for Ca, P and the ratioCa/C in reparative dentin compared with nor-mal dentin.

Normal calcification takes place in a pre-formed organic matrix produced by spe-cialised cells e.g. odontoblast and osteoblasts[1]. The original odontoblast layer after havingformed the primary dentin may becomereduced and in some teeth only a single layerwas visible. Hard tissue apposition along theroot canal walls is a slow normally occurringphysiological aging process. Thus the preva-lence of pulpal calcification in the primarydentition is expected to be lower than in thepermanent dentition. But also in the primarydentin the formation of reparative dentinseems to represent one of the earliest defencemechanisms of the tooth. It is well known thatthe rate of it may seem to be uncontrolledafter dental trauma [3, 7-8]. The onset ofreparative dentin formation can be moreclearly defined as the first possible momentsfor its formation to start is when few or largergroup of dentinal tubules are exposed toexternal irritants. Some of the teeth seemed tohave been subjected to milder injuries (sub-luxation) which seem to not affect the under-

lying odontoblasts as the more severe injuriesdo. If only some of them are destroyed the sur-vivors probably produce the reparative dentin.Reparative dentin differs from primary dentinand secondary dentin and it was thus easilydistinguished from these with the light micro-scope. It seems that the reparative dentin inthese cases looked more dentin-like. It wasmore regular and well-organised. It appearsthat the milder the injury is to dentin, thelower is the probability that reparative dentinwill be seen.

In the cases of moderate injuries (extrusiveand lateral luxation) that affect the blood sup-ply and cause cell death, the death of odonto-blasts can probably stimulate the underlyingcells to produce reparative dentin. The tissuedamage may produce localised metabolicchanges thereby promoting calcification. Thecalcifications here seem to be more irregular inpattern and sometimes had a more fibro-dentinal or bone structure. The number oftubules was less than in the primary dentinand the tubules had an irregular pattern. Theamount of reparative dentin formed pulpallyis probably well in correlation with theamount destroyed in the periphery and isprobably also related to the intensity, natureand duration of the external irritant. The dif-ferent types of reparative dentin are in accor-dance with earlier studies [3].

The condition of the pulp after dentaltrauma injuries has been evaluated in differ-

mate contact with the dentin. This calcificationcould be seen in types 2, 3 and 5 (Figure 3a). Inmost of the cases it seems that it has an inti-mate contact with the dentin as the interfacebetween the reparative dentin and normaldentin was rough and diffuse (Figure 3b). Thedeposition of calcified tissue on the dentinalwalls may strengthen the existing root struc-ture and may prevent resorptions on the rootsurface. This is in agreement with earlier stud-ies on teeth with pulp canal obliterationwhere very few cases with resorption werereported [3,7,8].

The chemical analyses of the reparativedentin showed a less well mineralized dentinwhich is in line with the differences in the mor-phological appearance. Even if the materialanalysed is limited in number, the values forthe ratio Ca/C in reparative dentin in particu-lar clearly point to a higher content of organicmatter compared with normal dentin. Onereason for the differences found could possi-bly be the activation of already-existing odon-toblasts as well as a the possible differentia-tion of new odontoblasts [10].

C O N C L U S I O N SThe present results show that reparativedentin is formed when pulp in primary teeth isexposed to subluxation and luxation. The var-ied morphology of the reparative dentin indi-cates that different stimuli lead to induction ofhard tissue forming cells which produce dif-ferent types of hard tissue. It looks like thereparative dentin formation is an important

protective function of the dental pulp. A dif-ference in the chemical composition of repar-ative and normal dentin was found for Ca, Pand the ratio Ca/C with lower values in repar-ative dentin.

R E F E R E N C E S1. Lindhe A. Dentin and Dentinogenesis. Boca Raton: CRC

Press, 1984.2. Appleton J, Williams J. R. Ultrastructural observations on

the calcification of human dental pulp. Calc. Tiss. Res.11:222-37, 1973.

3. Robertson, A. et al. Pulp calcifications in traumatizedprimary incisors. Eur. J. Oral Sci. 105:196-206, 1997.

4. Yaacob, H., Hamid, J. Pulpal calcifications in primary teeth. JPedodont. 1986:10:254-64.

5. Jacobsen, I., Sangnes, G. Traumatized primary anteriorteeth. Prognosis related to calcific reactions in the pulpcavity. Acta Odontol. Scand. 36:199-204, 1978.

6. Schröder, U. et al. Traumatized primary incisors- follow-upprogram based on frequency of periapical osteitis related totooth color. Swed. Dent. J. 1:95-98, 1977.

7. Robertson, A. et al. Incidence of pulp necosis subsequent topulp canal obliteration from trauma of permanent incisors.J. Endodont. 22:557-560, 1996.

8. Jacobsen, I., Kerekes, K. Long-term prognosis oftraumatized permanent anterior teeth showing calcifyingprocesses in the pulp cavity. Scand. J. Dent. 1977:85:588-98.

9. Andreasen, F. M. et al. Occurence of pulp canal obliterationafter luxation injuries in the permanent dentition.Endodont. Dent. Traumatol. 3:103-15, 1987.

10. Linde, A., Goldberg, M. Dentinogenesis. Crit. Rev. Oral Biol.Med. 4:679-728, 1993.

©2009 John Wiley & Sons, Ltd

ent studies. In clinical studies in permanentteeth it has been evaluated by electrical andthermal stimulation [7,8]. In contrast to clinicalstudies histological studies indicate that pulpalchanges often occur [3]. An adequate indica-tion of the vitality of pulp cells has been theirability to continue hard-tissue formation, a cri-terion widely used for functioning pulp tissuein post-traumatic roentgenologic evaluation[7,8]. However, relatively little experimentalevidence is available concerning the incidenceand reestablishment of the hard-tissue forma-tion in the pulp after a general trauma. Thefindings in this study confirm that dentinmatrix formation is a reliable determinant ofpulpal cell function after a general trauma tothe primary dentition. In no cases were inflam-matory infiltrates observed and the pulps werein good health. However, in this study therewere no teeth with severe injury as intrusiveluxations. Andreasen et al. [9] suggest that amoderate injury gives the highest frequencyof PCO and that a severe injury more oftenresults in pulp necrosis. The importance of avital pulp for the development of PCO is also inagreement with a higher frequency of PCOamong luxated teeth with open apices thanamong those with closed apices [9].

In the denticles, interglobular dentin wasfrequently observed and in some teeth incre-mental lines with alternating high and lowmineral content were seen. Indicating that ter-tiary dentin like other mineralized tissues issubject to biological rhythms during forma-tion. This is in agreement with earlier studies[2,4]. However, the factors involved in thedevelopment of the denticles are largelyunknown.

Diffuse calcifications were the most commoncalcification and it seems to be the simplestcalcification. This is in accordance with earlierstudies [4]. They occurred in all teeth and itseem that this type of calcification occur moreoften than the formation of denticles.

Tooth wear occurs during normal mastica-tion and attrition in the primary dentition.Pathologic wear, including abrasion and ero-sion may also take place. The reparative dentinformation is an important protective functionof the dental pulp. It is also a feature of pulpalhealing in response to injury and bacterialchanges. Thus it seems that it could be a pro-tection against bacterial invasion in the pri-mary teeth, which usually demonstrate fewcomplications and symptoms after attrition.Furthermore, very few complications arereported after crown fracture without pulpexposure. These fractures may often only betreated by grinding sharp edges. Formation ofreparative dentin and obturation of dentinaltubules are biological responses that may com-pensate for the loss of tissue especially in theprimary teeth. The mineralization seemed tostart in the incisal region and the central partis the last part to be mineralized. This processwill increase the thickness of the remainingdentin and decrease the risk that the pulp willbe affected by noxious influences. The deposi-tion of calcified tissue in the pulp cavity mayprevent bacterial invasion to the pulp.

The calcified tissue may be diffuse or in inti-

MICROSCOPY AND ANALYSIS MAY 200916

C O P Ca Ca/P Ca/C

N RD N RD N RD N RD N RD N RD

4.02 4.47 40.01 41.22 17.01 16.70 39.19 37.59 2.30 2.24 9.93 8.66

* * **

Tooth number 52 51 61 62 82 72

Number 2 6 8 2 2 2

Table 2:Median values for C, O, P, Ca, Ca/P and Ca/C in normal and reparative dentin (N=normal dentin; RD=reparative dentin; *=p<0.05; **=p<0.01].

Table 1: Type and number of the analyzed primary teeth.

Figure 5: Photomicrograph of free denticles and diffuse calcifications in the pulp cavity. Scale bar=100 µm.