socket healing

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Modeling and remodeling ofhuman extraction sockets

Trombelli L, Farina R, Marzola A, Bozzi L, Liljenberg B, Lindhe J. Modeling andremodeling of human extraction sockets. J Clin Periodontol 2008; 35: 630639. doi:10.1111/j.1600-051X.2008.01246.x.

Abstract

Introduction: The available studies on extraction wound repair in humans are

affected by significant limitations and have failed to evaluate tissue alterations

occurring in all compartments of the hard tissue defect.Aim: To monitor during a 6-month period the healing of human extraction socketsand include a semi-quantitative analysis of tissues and cell populations involved invarious stages of the processes of modeling/remodeling.

Material and Methods: Twenty-seven biopsies, representative of the early (24weeks, n510), intermediate (68 weeks, n5 6), and late phase (1224 weeks,n5 11)of healing, were collected and analysed.

Results: Granulation tissue that was present in comparatively large amounts in theearly healing phase of socket healing, was in the interval between the early and

intermediate observation phase replaced with provisional matrix and woven bone. Thedensity of vascular structures and macrophages slowly decreased from 2 to 4 weeksover time. The presence of osteoblasts peaked at 68 weeks and remained almoststable thereafter; a small number of osteoclasts were present in a few specimens at

each observation interval.

Conclusions: The present findings demonstrated that great variability exists in manwith respect to hard tissue formation within extraction sockets. Thus, whereas aprovisional connective tissue consistently forms within the first weeks of healing, theinterval during which mineralized bone is laid down is much less predictable.

Key words: alveolar ridge; bone healing;

histomorphometric analysis; tissue modeling/remodeling; tooth extraction

Accepted for publication 6 April 2008

The alveolar process forms during tootheruption (Schroeder 1986) and under-goes atrophy after the loss of single ormultiple teeth (e.g. Pietrokovski &Massler 1967, Johnson 1969, Schroppet al. 2003, Pietrokovski et al. 2007). As

a rule, the resorption of the buccalcompartment of the ridge after toothremoval is more pronounced than the

amount of tissue that is lost in thelingual/palatal portion. The alterationof the ridge occurs concomitantly withthe healing of the soft and hard tissuewound but the process of remodelingmay continue also after the termination

of de novo bone formation in thesocket(s) (Schropp et al. 2003).Processes involved in the healing of

an extraction socket have been studiedin different animal models (e.g. Kubokiet al. 1988, Lin et al. 1994, Lekic et al.2001, Cardaropoli et al. 2003, 2005,Kanayama et al. 2003, Sato & Takeda2007). In a recent study using a dogmodel, Cardaropoli et al. (2003)explored the healing of the coronal,central and apical compartments of freshextraction sockets during a 6-monthinterval. The authors observed that in

this model, the socket already after 4weeks of healing was filled with wovenbone and that after 2 months this imma-ture bone had been replaced with lamel-lar bone and marrow. Concomitant withthese intra-alveolar healing events, the

buccal wall of the socket underwentmarked resorption. Thus, in comparisonto the lingual wall of the socket, theheight of its buccal counterpart wasreduced on the average 2.5 mm (Araujo& Lindhe 2005).

Tissue formation after tooth extrac-tion was also studied in human models(Claflin 1936, Mangos 1941, Christopher1942, Amler et al. 1960, 1964, 1969,Boyne 1966, Evian et al. 1982). Amler(1969) examined new tissue formationin the marginal portion of extractionsites from human volunteers at healing

Leonardo Trombelli1

, RobertoFarina1, Andrea Marzola2, Leopoldo

Bozzi3, Birgitta Liljenberg4 and

Jan Lindhe4

1Research Centre for the Study of

Periodontal Diseases, University of Ferrara,

Ferrara, Italy; 2Department of Experimental

and Diagnostic Medicine, Section of

Histologic and Citologic Anatomy, University

of Ferrara, Ferrara, Italy; 3Private Practice,

Comacchio, Italy; 4Department of

Periodontology, Faculty of Odontology, The

Sahlgrenska Academy at Goteborg

University, Goteborg, Sweden

Conflict of interest and source offunding statement

The authors declare that they have no

conflict of interest.This study was supported by the ResearchCentre for the Study of Periodontal Dis-eases, University of Ferrara, Italy, and by a

grant from the University of Ferrara, Italy(STAMINA).

J Clin Periodontol 2008; 35: 630639 doi: 10.1111/j.1600-051X.2008.01246.x

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intervals extending from 2 to 32 days.He concluded that the blood clot thatinitially filled the entrance of the socketwas first replaced with granulation tis-sue. After 1 week of tissue modeling,osteoid formation had begun and afterabout 6 weeks, the marginal portion of

the socket harboured islands of imma-ture woven bone. However, the humanstudies cited above (i) included tissuesamples from systemically diseasedindividuals or even cadavers (Claflin1936, Mangos 1941), (ii) had an unclearexperimental protocol (Christopher1942), (iii) evaluated only few tissuesamples and/or (iv) short observationintervals (Amler et al. 1960, Boyne1966, Evian et al. 1982).

On the basis of the above concerns, astudy was designed to (i) monitor thehealing of human extraction sockets and

(ii) include a semi-quantitative analysisof tissues and cell populations involvedin various stages of the healing process.

Material and Methods

Patient and site selection

Subjects were considered eligible for thestudy if they were X18-years old and ifthey did not suffer from systemic dis-ease. Pregnant or lactating women, andsubjects taking (past or currently) drugsinfluencing bone metabolism were

excluded. The study was approved bythe Ethical Committee of the Universityof Ferrara. All included subjectsprovided an informed consent beforeparticipation.

The following tooth sites were con-sidered: single rooted-teeth, molars orpremolars with fused roots or singleroots of molars. Biopsy was consistentlyperformed in conjunction with perio-dontal surgery or implant placement inthe extraction site. Teeth with (i) peria-pical lesion were excluded as well as (ii)sites with limited depth (o5 mm) of the

remaining sockets after tooth removal.

Extraction and post-extraction protocol

Full thickness buccal and lingual muco-periosteal flaps were elevated. The toothwas carefully removed with the use ofelevators. No ostectomy or osteoplastywas performed. No graft or membranedevice was used to augment the site.Flaps were sutured with 5/0 silk orVycrils suture (ETHICON, Johnson &Johnson, Cincinnati, OH, USA). Thewound was left to heal for primary

intention. When needed, partial thick-ness dissection in the most apical por-tion of the flap was performed in orderto coronally advance the flap. Localantimicrobial therapy (chlorhexidine0.12% mouthrinse; 10 ml for 60 s t.i.d.for 7 days) was prescribed. Sutures were

removed 1 week postoperatively. If alocal complication (e.g. rupture of softtissue wound) occurred during healingand/or systemic antibiotic therapy had tobe prescribed, the site was excludedfrom the study.

Localization of the extraction site

Before tooth extraction, an alginateimpression (Xantalgins; Heraeus KulzerS.r.l., Milan, Italy) was obtained of theexperimental area. On a cast model, aresin/silicon stent was prepared and

adapted on the dentition adjacent to thetooth to be removed. A canal parallelwith the long axis of the tooth wasprepared in the stent. This allowed theoperator to properly identify the socketsite during biopsy.

Immediately after tooth extraction,the distance from the stent to the bottomof the socket was measured. This wasmade with a UNC-15 periodontal probe.This recording was used in order toensure that, at the time of biopsy, thetrephine bur would collect the newlyformed tissue only.

Collection and storage of the specimens

The timing of the biopsy procedure wasselected in accordance with the clinicalneed for the surgical procedure (i.e.periodontal surgery or implant position-ing) involving the extraction socket. Atthe time of biopsy retrieval, local infil-tration of anaesthetic was administered.Whenever the concomitant surgical pro-cedure required a full-thickness flap, acrestal incision was performed and amucoperiosteal flap was elevated on

both buccal and lingual/palatal sidewith a microsurgical periosteal elevator(P-TROM; Hu-Friedy, Chicago, IL,USA). When this was not the case(e.g. implant positioning with a flaplessapproach), the soft tissues overlying theextraction socket were included in thebiopsy. The tissue specimens were col-lected with a trephine bur (internaldiameter 2 mm; Hu-Friedy) from thehealing sockets. The depth of the tre-phine bur insertion was related to themeasurements previously made by thestent (see Localization of the extrac-

tion site). The tissue was obtainedfrom the centre of the socket, with theinsertion axis of the trephine kept par-allel to the long axis of the adjacenttooth. The apical portion of the speci-men was marked with a fine indeliblechine pen.

Histological and immunohistochemical

processing

The specimens were immediatelyplaced in a 10% formaldehyde fixative,decalcified in ethylene diaminetetraaceticacid dehydrated in increasing con-centrations of ethanol, embedded inparaffin and cut in the sagittal planewith the microtome set at 5 mm. From

each biopsy, between 10 and 20 sectionsrepresenting the central part of the sock-et were selected for histological and

immune-histochemical examinations.One section was stained in haema-toxylineeosine, and examined in amicroscope (Leitzs DM-RBE Micro-scope; Leica, Wetzlar, Germany)equipped with an image system (Q-500MCs; Leica).

The total cross-section area of thetissue sample was outlined and the sizedetermined by planimetry. Subse-quently, the areas (proportions of thecross-section area) occupied by (1) clot(erythrocytes, leukocytes embedded in afibrin network), (2) granulation tissue

(rich in newly formed vascular struc-tures, abundance of inflammatory cellssuch as neutrophils, macrophages, lym-phocytes as well as erythrocytes), (3)provisional matrix (densely packedmesenchymal cells, collagen fibres andvessels but no or only scattered inflam-matory cells), (4) woven bone (finger-like projections of immature boneembedded in a primary spongiosa) and(5) lamellar bone and marrow, werecalculated.

A series of paraffin sections were de-paraffinized, treated with TE-buffer (pH

9) or Proteinase K (Dakacytomation,Glostrup, Denmark) and washed inTris-buffered saline (TBS) for 3 min.Endogenous peroxidase activity wasblocked with a peroxidase solutionfor 10 min. and subsequently rinsedfor 3 min. with TBS. A panel of mono-clonal antibodies diluted in TBSwas applied for 30 min. and washed inTBS for 2 3 min. The sections wereincubated with a peroxidase labelledpolymer for 30min. and washed inTBS for 3 3 min. Diaminobenzidinetetrahydrochloride (DAB) was used as

Healing of human post-extraction sockets 631

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substrate/chromogen (EnVision1s

System-HRP (DAP) Kit; Dakocytoma-tion). The sections were rinsed in TBSand distilled water, counterstained inMayers haematoxyline stain and

mounted. For each antibody tested, onesection was incubated in serum andserved as a negative control.

The various antibodies were used toidentify vascular (endothelial cells)structures (CD31), osteoblasts (BMP-7,Osteocalcin) osteoclasts (RANK/TNFRSF 11A) and macrophages, giantcells (CD68). The monoclonal antibo-dies used, their isotypes and dilutionsare presented in Table 1.

Semi-quantitation

The morphometric assessment onCD31-stained sections was conductedaccording to methods described earlier(Schroeder & Munzel-Pedrazzoli 1973).Briefly, the cross-section area of thetissue samples was determined (1.6)and vascular structures were subse-quently identified and enumerated(10). The numerical density of vascu-lar units per area unit (mm2) of availabletissue was then calculated.

A semi-quantitative analysis of BMP-7-, osteocalcin- and CD68-positive cellswas conducted. For each investigated

protein/cell type, a score ranging from0 (absent) to 4 (large amounts) wasdetermined.

BMP-7 and osteocalcin

BMP-7 and osteocalcin scores wererelated to the percentage of the periph-ery of the woven bone projections thatshowed the presence of BMP-7- andosteocalcin-stained cells, respectively.

1 o10%2 425% buto40%

3 450% buto75%4 480%

CD68CD68 score was related to the numberof CD68-stained cells in the entire tissuesample.

1 o20 cells2 3050 cells3 50100 cells4 4200 cells

All the histologic evaluations and histo-morphometric measurements were car-ried out by a single trained andcalibrated examiner (B. L.). Data were

expressed as mean

SD.

Results

Twenty-four patients (10 males and 14females, range 3472 years) wereincluded. Twenty-two patients contribu-ted with one, one patient with two and onepatient with three extraction sites. All thebiopsy procedures were performed with-out any major intra-operative complica-tions. Occasionally, the sampled tissuefragmented into two to three separate

pieces during biopsy harvesting, espe-cially when newly formed hard tissuewas present in the extraction socket.When this was the case, all the pieceswere retrieved, oriented, stored togetherand analysed as a single sample.

The location of the extraction siteswas as follows: 1 lateral incisor, 1canine, 19 premolars, 6 molars. Twelveteeth were obtained from the upper jawand fifteen from the lower jaw. Teethwere extracted due to caries (n5 8),vertical root fracture (n5 9), failure ofendodontic therapy (n5 3), periodontal

disease (n5 5), or for prosthetic reasons(n5 2).

Of the 27 biopsies that were availablefor analysis, 10 were sampled to repre-sent early healing (24 weeks), sixintermediate (68 weeks), and 11 a laterphase (1224 weeks) of healing.

Gross morphological characteristics of

the tissue samples

In most specimens examined, it waspossible to distinguish between differenttypes of tissues. Thus, in some regionsthe biopsy could disclose features typi-cal of granulation tissue whereas inother regions of the same specimen,tissue modeling was more advanced

and areas including provisional matrixand/or woven bone could be identified.

Granulation tissue was characterized

by the presence of large numbers ofvascular structures in a connective tissuecomprising mesenchymal cells and infil-trates of leukocytes (neutrophilic leuko-cytes, macrophages and lymphocytes)(Fig. 1). The inflammatory cells weremainly present in peri-vascular compart-ments (Fig. 2) but occurred also in areasmore distant to vascular structures.

Provisional matrix contained denselypacked mesenchymal cells present in acollagen-rich connective tissue matrix(Fig. 3). Vascular structures (Fig. 4)were abundant but only few infiltrates

of leukocytes (macrophages and lym-phocytes) could be observed. Osteo-blasts (osteocalcin-stained cells)occurred in peri-vascular locations.

Woven bone occurred as fingerlikeprojections of mineralized tissue (Fig.5) in a connective tissue matrix. Theridges of woven bone were lined withosteoblasts and contained large numbersof osteocytes (Fig. 6). The trabeculaeof mineralized bone occurred in thevicinity of, often surrounding, one orseveral vascular structures (Fig. 7).Osteoclasts (RANK/TNFRSF 11A-

positive cells) occurred in discrete areasat the surface of woven bone trabeculaeand were consistently present in How-ships lacunae (Fig. 8).

Only one section (12 weeks of heal-ing) (Fig. 9) comprised lamellar boneand marrow. The mineralized boneharboured secondary osteons whereasthe bone marrow was rich in vessels,adipocytes, mesenchymal cells andinflammatory cells (macrophages, lym-phocytes). Osteoclasts were present inseveral areas at the surface of the miner-alized bone.

Table 1. Isotypes and dilutions of the monoclonal antibodies used for immuno-histochemical

stainings

Antibodies (clone) Specificity Dilutions Isotype Source

CD 31 (JC70A) Endothelial cell 1:20 IgG1 Dakocytomation,

Glostrup DenmarkBMP7 (164313) BMP7 1:20 IgG1 R&D Systems, Europe Ltd

Oxon, UK

Osteocalcin (190125) Osteoblast 1:10 IgG1 R&D Systems, Europe LtdCD68 Macrophage 1:20 IgG1 Diagnostic Biosystems,

Pleasanton, CA, USA

RANK/TNFRSF11A(80707)

Osteoclast 1:20 IgG2A R&D Systems, Europe Ltd

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Histomorphometric measurements

The composition of each tissue sampleis shown in Table 2. Only in samplesrepresenting the early interval of healing(24 weeks) could few, scattered ery-throcytes be observed in various parts of

the sections. However, no definitive clotformation could be distinguished.

Granulation tissue

In 4 out of 10 samples representing theearly healing interval, granulation tissue

could be observed (Table 2). In thesespecimens, the richly vascularized, hea-vy infiltrated tissue occupied between67% and 100% of the samples exam-ined. Six of the sections contained nogranulation tissue. The overall meanarea occupied by granulation tissue in

this healing interval was 35.9

47.2%(Fig. 10).Only one of the six biopsies repre-

senting the intermediate healing interval(68 weeks; Table 2) contained a typicalgranulation tissue, whereas no specimenin the 1224 week interval harbouredthis type of immature tissue.

Provisional matrix

A tissue rich in mesenchymal cells,collagen fibres and vascular structureswas found in 7 out of 10 specimens from

the early healing interval. In these speci-mens, the provisional matrix occupiedbetween 33% and 100% of the tissueexamined with mean value 57.2 44.2% (Table 2). All sections from theintermediate healing period harboureddifferently large zones (between 27%and 98%; mean value 62.2 23.8%)of provisional matrix. Ten out of elevenbiopsies from the late healing intervalcontained a typical provisional matrixtissue. In these sections, between 35%and 92% of the area examined containedthis type of tissue with mean value

58.5

24.5%.

Woven bone and lamellar bone

Areas occupied by woven bone could beobserved already in specimens from the24 week interval. Thus, in 6 out of 10biopsies (Table 2) fingerlike projections

of newly formed bone could be identi-fied. When present in the sections, thisimmature bone occupied between 2%and 34% of the tissue area (mean6.9 10.5%; Fig. 10).

In practically all specimens represent-

ing the intermediate and late healinginterval, woven bone was present andoccupied between 2% and 73% of thetissue examined (mean value 68weeks: 34.0 24.6%; 1224 weeks:32.4 18.4%). Lamellar bone and mar-row was found in only one biopsy andthis tissue represented the 1224 weekhealing interval (Table 2).

Immunohistochemical analysis

Table 3 shows the presence of positivecells for each staining.

Fig.1. A biopsy obtained after 3 weeks of healing. The tissue is rich in vessels, fibroblastsand inflammatory cells and is characterized as granulation tissue. Original magnification2.5. Inset: large amounts of inflammatory cells can be observed immediately beneath theoral epithelium. Original magnification 10. H & E stain.

Fig.2. Detail from Fig. 1 illustrating vascular structures that have been stained with CD 31.Note the large amount of vessels of varying diameters including several vascular sprouts(arrows). Original magnification 10.




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The number of CD31 positive cellsper area unit (mm2) was 33.0 12.2 at24 weeks, 27.2 5.6 at 68 weeks and19.0 8.8 at 1224 weeks.

BMP-7 score shifted from 1.5 0.6at 24 weeks to 2.8 1.0 at 68 weeks,and 2.1 1.2 at 1224 weeks.

Osteocalcin score shifted from1.4 0.6 at 24 weeks to 2.5 1.0 at

68 weeks, and 1.9 0.8 at 1224weeks.

CD68 score shifted from 2.8 1.0 at24 weeks to 2.1 1.1 at 68 weeks,and 1.1 0.4 at 1224 weeks.

RANK/TNFRSF 11A-positive cellswere detected in low numbers (one totwo cells) in only 3 out of 10 specimensat 24 weeks. At 68 weeks, only few

(one to three cells) osteoclasts werefound in five out of the six samples. Inone sample, however, a large number ofosteoclasts (410 cells) were stained. Inspecimens representing 1224 weeks,few osteoclasts were found in 6 out of11 samples; i.e. five were negative.

Discussion

The overall findings from the presentstudy demonstrated that the healing ofan extraction socket, as disclosed in ourpanel of subjects, followed a patternsimilar to that previously describedfrom histological examinations of tis-sues harvested from socket sites in man

and animals (for review see Chen et al.2004, Cardaropoli 2006). The process ofbone modeling/remodeling described in

the present biopsy samples has severalfeatures in common with intra-membra-neous bone formation (Whistson 1994)and bone formation that occurs in mem-brane protected defects in the alveolarridge (Schenk et al. 1994). Data alsodisclosed that (i) the rate of healingvaried markedly between subjects; (ii)the process of remodeling, i.e. the repla-cement of woven bone with lamellarbone and marrow was slow. Only 1 ofthe 11 specimens representing 1224weeks of healing comprised maturebone, i.e. lamellar bone and marrow;

(iii) density of vascular structures washigh at 24 and 68 weeks and tended todecrease in the 1224 week specimens;(iv) presence of osteoblasts peaked at 68 weeks and remained almost stablethereafter; (v) macrophages slowlydecreased from 2 to 4 weeks over timeand (vi) osteoclasts were present inonly few specimens at each observationinterval.

Immediately after tooth extraction, thesocket fills with blood and clot formationoccurs (Amler 1969, Cardaropoli et al.2003). In the present material, erythro-

cytes scattered in between mesenchymalcells were frequently observed in thetissue from biopsies representing 24weeks, although typical clot formations(erythrocytes, platelets and leukocytesentrapped in a dense fibrin network)could not be observed in any of thetissues examined. This finding is inagreement with data presented by Amleret al. (1960) and Amler (1969) whodocumented that the blood clot that firstfilled the socket space was almostentirely remodeled within the firstweek after tooth removal. Furthermore,

Fig.3. A biopsy obtained after 4 weeks of healing. The provisional matrix comprisesmesenchymal cells densely packed fibres and vessels. Only few inflammatory cells can beobserved. Original magnification 2.5. Inset: at the lateral border of the section, minute areasof woven bone formation can be identified. Original magnification 10. H & E stain.

Fig.4. Detail from Fig. 3 illustrating vascular structures that have been stained with CD 31.Original magnification 10.




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most of the granulation tissue thatoccurred in samples representing theearly phase (24 weeks) of healing,had in the interval between the earlyand intermediate healing phase (68

weeks) apparently been replaced withprovisional matrix and woven bone.This observation corroborates findingsby Amler et al. (1960) and Evian et al.(1982) who reported that between the

first and third week of socket healingthere was a progressive replacement ofgranulation tissue with connective tis-sue. Provisional matrix and woven bonealso dominated in biopsies from the latephase of healing (1224 weeks) of thecurrent study, and only one specimen

(representing 12 weeks) included sub-stantial amounts of lamellar bone andmarrow. In other words, whereas tissuemodeling in the human extraction sitesappeared to be a comparatively fastprocess, remodeling of the newlyformed hard tissue was seemingly slow.

The observation that bone remodelingof extraction sites in man is a slowprocess is in many respects consistentwith data presented by Schropp et al.(2003). They used clinical and radio-graphic means to monitor tissue altera-tions within and outside extraction sites

in the premolar and molar regions of 46patients. The data described by theauthors disclosed that the dimensionsof the extraction sites were markedlyreduced after tooth removal. Thus, at3 months 430% and at 12 months 450%of the buccallingual/palatal width ofthe marginal portion of the ridge waslost after tooth removal. Moreover, itwas noted that large amounts of miner-alized tissue formed in the sockets dur-ing the first 6 months whereas in theinterval between 6 and 12 months, theamount of newly formed mineralized

tissue was substantially reduced (wovenbone was replaced with lamellar boneand marrow). The findings by Schroppet al. (2003) thus demonstrate that heal-ing of an extraction site in man proceedswith the resorption of the marginalsocket walls as well as with varyingdegree of hard tissue fill within thesocket. It is important to emphasizethat the biopsy technique used in thecurrent study limited the analysis to theintra-alveolar events.

In a recent publication, Chen et al.(2004) reported on osseous regeneration

in human extraction sockets andincluded four studies with histologicdocumentation in their review (Amleret al. 1960, 1969, Boyne 1966, Evianet al. 1982). Amler (1969) claimed thatafter about 40 days of healing two-thirdof the sockets were filled with miner-alized bone, whereas Evian et al. (1982)stated that the sockets were completelyfilled with bone after 10 weeks of heal-ing. The findings from the current studycould only in part corroborate the abovefindings. Thus, in specimens obtainedbetween 6 and 8 weeks woven bone

Fig.5. Decalcified section obtained from a biopsy sampled after 6 weeks of healing. Note thepresence of trabeculae of immaturewoven bone that occur in a cell and fibre-rich provisionalmatrix. Original magnification 2.5. Inset: the projections of woven bone are enclosingvascular structures to form primary osteons. Original magnification 10. H & E stain.

Fig.6. Detail from Fig. 5 illustrating osteoblasts that line the newly formed woven bone aswell as osteocytes within the immature bone. Osteocalcin staining. Original magnification20.




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occupied about 35% of the tissue sample,whereas after 12 and 24 weeks of woundhealing only about 41% of the biopsy

harvested from the socket comprisedmineralized bone.

Various aspects of healing of extrac-tion sockets were studied in the dogmodel (for review see Chen et al.2004, Cardaropoli 2006). In such care-fully monitored animal experiments, (i)the teeth were carefully removed fromperiodontally non-compromised sitesand (ii) healing was analysed in biopsiescontaining not only the hard tissuedefect but in addition the surroundingbone walls (mesial, distal, buccal, lin-gual). In such experiments, new tissue

formation in various parts of the socketfollowed a well-defined course. Thus,the clot that formed immediately after

tooth removal was in the first few daysconsistently replaced with granulationtissue that already after 1 week in parthad been replaced with a cell richprovisional matrix. After 2 weeks ofhealing, large portions of the apicaland lateral compartments of the socketwere occupied by woven bone and after4 weeks, the process of modeling wasadvanced. Hence, in the intervalbetween 4 and 8 weeks, the newlyformed immature woven bone wasreplaced with lamellar bone and mar-row. In the tissue samples harvested

from the human extraction sitesincluded in the current study, this ratherdistinct pattern of tissue modeling/remo-deling was difficult to identify. In con-trast, the results from the histologicalexaminations revealed great variationbetween samples with respect to tissue

formation and maturation. The reasonfor this variation is presently not under-stood but may be linked to subject-related factors, pre-existing damage tothe tooth and its supporting tissues (rea-son for tooth extraction), traumainflicted to the tissues in conjunctionwith tooth removal, the size of thehard tissue defect, etc.

In our study, immunohistochemicalstainings were used to investigate thetime-dependent changes in density ofthe main cell types involved in post-extraction wound healing. The density

of endothelial cells, assumed as corre-sponding to the number of CD31-positive cells, was decreased at 1224weeks with respect to 24 weeks and68 week observations. This findingcan be explained by the progressiveremodeling of the highly vascularizedgranulation tissue and its subsequentreplacement by provisional matrix.

The histomorphometric analysisincluded the use of antibodies againstBMP-7 and osteocalcin. For these mea-surements, only positive cells lining thenewly formed trabeculae of woven bone

were considered in order to limit thestudy to the cells directly involved in thebone forming and remodeling process.For both antibodies, the density of posi-tive cells increased from 24 weeks to68 weeks and tended to decrease at1224 weeks. The increase in BMP-7positive cells between the early andintermediate healing phase can be attrib-uted to an increased bone modeling andremodeling activity, leading to thedeposition of woven bone from provi-sional matrix. In this respect, previousstudies demonstrated that BMP-7 is

highly expressed during the early stagesof bone repair after a fracture (Spectoret al. 2001, Kloen et al. 2003). Thisprotein continues to be expressed duringthe process of bone remodeling, albeitless prominently, and the return ofBMP-7 density to baseline value coin-cides with the histological appearance ofmature lamellar bone (Spector et al.2001). In this respect, the limited pre-sence of lamellar bone in the 1224week specimens observed in the currentstudy was associated with a tendency fordecrease in BMP-7 levels.

Fig.7. Detail from Fig. 5. The tissue was stained with CD 31 and illustrates vascularstructures that reside in the centre of primary osteons. Original magnification 20.

Fig.8. Detail from Fig 5. Note the presence of osteoclasts on the surface of newly formedbone. Stain: Rank/TNFRSF 11A. Original magnification 40.




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The osteocalcin expression paralleledBMP-7 expression during the entireobservation interval. Osteocalcin is anosteoblast-specific marker, being spa-

tially confined to osteoblasts activelylaying down new osteoid or remodelingbone (Paccione et al. 2001). Because ofits progressive accumulation in the

maturing bone matrix, osteocalcinrepresents a late marker of bone forma-tion. In fact, a greater expression ofosteocalcin-positive cells can beobserved in the mature bone matrixthan in newly formed bone (Ivanovskiet al. 2000, Reinhardt et al. 2005). It was

demonstrated that osteocalcin, used as amarker to define alveolar bone turn-overin humans, was negatively correlatedwith increasing radiographic density ofexperimentally produced bone cores, i.e.the higher the osteocalcin expression theless dense was the amount of newmineralized bone (Reinhardt et al.2004).

For obvious reasons, the current studyhad a cross-sectional design. Mostpatients contributed with only onebiopsy from one healing interval. Thus,it can only be assumed that an immature

tissue identified in biopsies from 2 to 4weeks interval would have matured toform bone if continued healing wouldhave been allowed. Furthermore, cellsidentified as osteoblasts or pre-osteoblasts in sections stained with monoclonalantibodies for osteocalcin and BMP-7 varied in numbers from one biopsy tothe next (Table 3) but reached peakvalues in sections representing 68weeks of healing. In such sections,however, a large number of typicalmesenchymal cells were negative forthe monoclonal antibodies used. This

may indicate that such cells were notin an active bone-forming phase orrepresented populations of cells notinvolved in the formation of bone. It isa well-known fact that cells that re-populate a wound determine the natureof the tissues formed (Melcher 1976).Thus, it cannot be taken for granted thattissue portions in the present biopsysample were occupied by provisionalmatrix would become replaced withwoven bone.

In our study, osteoclasts, whendetected, were present in low numbers

(one to three cells) at each observationinterval. In a dog study by Cardaropoliet al. (2003), a substantial remodelingactivity with an increased number ofosteoclasts was observed at 1 monthafter tooth extraction, leading to adecrease in the area occupied by miner-alized bone. In humans, a similar remo-deling activity was observed not before6 months after tooth removal, i.e. at alonger observation interval with respectto our study (Schropp et al. 2003).Therefore, it may be hypothesized thatour study, due to earlier observation

Fig.9. Biopsy obtained from an extraction wound representing 12 weeks of healing. The tissue

comprises more mature bone; woven bone and lamellar bone that reside in a non-mineralizedmatrix. Original magnification 2.5. H & E stain. Inset A: note the presence of secondaryosteons in lamellar bone. Original magnification 40. Inset B: large number of inflammatorycells among adipocytes in the newly regenerated bone marrow. Original magnification 40.

Table 2. Composition (percentage) of each sample with respect to clot, granulation tissue,

provisional connective tissue, woven bone and lamellar bone/bone marrow

Sample

no.

Clot

(%)

Granulation

tissue (%)

Provisional connective

tissue (%)

Woven bone

(%)

Lamellar bone/bone

marrow (%)

24 weeks

#1 0 100 0 0 0

#2 0 92 0 8 0#3 0 0 89 11 0

#4 0 0 89 11 0

#5 0 0 97 3 0#6 0 0 100 0 0#7 0 0 66 34 0

#8 0 100 0 0 0

#9 0 67 33 0 0#10 0 0 98 2 0

68 weeks

#1 0 0 27 73 0

#2 0 23 57 20 0#3 0 0 53 47 0

#4 0 0 76 24 0#5 0 0 62 38 0

#6 0 0 98 2 0

1224 weeks

#1 0 0 52 48 0#2 0 0 0 0 100

#3 0 0 92 8 0

#4 0 0 84 16 0#5 0 0 67 33 0

#6 0 0 35 65 0

#7 0 0 64 36 0

#8 0 0 58 42 0#9 0 0 60 40 0

#10 0 0 64 36 0

#11 0 0 68 32 0




9/10

intervals, could have anticipated thisremodeling phase.

The present findings demonstratedthat great variability exists in man withrespect to hard tissue formation withinextraction sockets. Thus, whereas a pro-visional connective tissue consistently

forms within the first weeks of healing,the interval during which mineralizedbone is laid down is much less predict-able. Despite the high heterogeneity thatwas observed in the composition of thespecimens within each observationinterval, each tissue component shows

a well-defined tendency to change overtime. Apparently, the bone organizationand architecture was not completed at24 weeks after tooth extraction.

Acknowledgements

This study was supported by theResearch Centre for the Study of Perio-dontal Diseases, University of Ferrara,Italy, by a grant from the University ofFerrara, Italy (STAMINA).

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Fig. 10. Distribution (mean %) of the tissue components (clot, granulation tissue, provisionalconnective tissue, woven bone, lamellar bone/bone marrow), calculated on all samples foreach observation interval. The standard deviation is shown in parenthesis.

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#7 14.8 1.5 2.0 Not available#8 13.7 1.0 2.0 1.5

#9 26.0 2.0 3.0 1.5

#10 17.8 3.0 2.0 Not available

#11 10.6 2.0 1.0 1.0Mean 19.0 2.1 1.9 1.1

SD 8.8 1.2 0.8 0.4




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Address:

Dr. Leonardo Trombelli

Research Center for the Study of Periodontal

Diseases

University of Ferrara

Corso Giovecca 203

44100 Ferrara

Italy

E-mail: [email protected]

Clinical Relevance

Scientific rationale for the study:Studies on post-extraction woundrepair are affected by significant lim-itations and do not evaluate all com-partments of the bone defect.Principal findings: The process ofremodeling, i.e. the replacement of

woven bone with lamellar bone andmarrow, is slow. Limited morpholo-gical alterations seem to occurbetween 68 and 1224 weeks ofhealing. High inter-individual het-erogeneity was noted within eachobservation interval.

Practical implications: The greatvariability observed in the rate ofnew bone formation from patient topatient may be of clinical relevancewhen to determine the most suitabletime for implant placement in post-extraction sockets.



socket healing

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