the potential for vertical bone regeneration via maxillary...

The potential for vertical boneregeneration via maxillaryperiosteal elevationMouraret S, von Kaeppler E, Bardet C, Hunter DJ, Chaussain C, Bouchard P,Helms JA. The potential for vertical bone regeneration via maxillary periostealelevation. J Clin Periodontol 2014; 41: 1170–1177. doi: 10.1111/jcpe.12310.

AbstractBackground: While many studies have been performed on the characteristics andregenerative capacity of long bone periosteum, the craniofacial periosteumremains poorly understood.Aim: The aim of this study was to investigate the potential for a maxillary perios-teum tunnelling procedure to induce vertical alveolar bone regeneration.Materials and Methods: We employed a murine injury model that activates skele-tal stem cells in the periosteum without overtly damaging the underlying corticalbone, preserving the integrity of the long bone and maxilla, and avoiding theintroduction of pathological motion at the injury site. Further, we introduced acollagen sponge to serve as a scaffold, providing the necessary space for verticalbone regeneration.Results: Periosteal elevation alone resulted in bone formation in the tibia anddelayed bone resorption in the maxilla. With the presence of the collagen sponge,new bone formation occurred in the maxilla.Conclusions: Periosteal response to injury varies with anatomical location, so con-clusions from long bone studies should not be extrapolated for craniofacial appli-cations. Murine maxillary periosteum has the osteogenic potential to inducevertical alveolar bone regeneration.

Sylvain Mouraret1,2,†, Erickavon Kaeppler1,†, Claire Bardet1,3,

Daniel J. Hunter1, CatherineChaussain3, Philippe Bouchard2 and

Jill A. Helms1

1Division of Plastic and Reconstructive

Surgery, Department of Surgery, Stanford

School of Medicine, Stanford, CA, USA;2Department of Periodontology, Service of

Odontology, Rothschild Hospital, AP-HP,

Paris 7 – Denis, Diderot University, U.F.R. of

Odontology, Paris, France; 3Dental School

University Paris Descartes Sorbonne Paris

Cit�e, Montrouge, France

†Contributed equally to the work.

Key words: collagen sponge; periosteum;

vertical bone regeneration

Accepted for publication 8 September 2014

The periosteum is a specialized, vas-cularized connective tissue anchoredto the surface of bone, and is gener-ally considered a reservoir ofundifferentiated, multi-potent mesen-chymal cells. The periosteum has

two distinct layers: the outerfibrous layer containing fibroblasts,nerves, vessels, and Sharpey’s fibres(Simon et al. 2003, Al-Qtaitat et al.2010) and the inner cambium layercontaining the osteoprogenitors(Siems et al. 2012). In vivo, theseperiosteal stem/progenitor cells areable to differentiate into chondro-genic and osteogenic lineages; invitro, they can be induced to differ-entiate into adipogenic and myo-genic lineages as well (Malizos &Papatheodorou 2005, Siems et al.2012).

Duhamel’s 1739 “silver ringexperiment” was considered the firstscientific demonstration of the osteo-genic capacity of the periosteum(Macewen 1907). Since those experi-ments in the early 18th century, theperiosteum has been under contin-ued investigation, with the goalbeing to harness its bone-formingpotential. For example, in severalsurgical orthopaedic techniques,including distraction osteogenesis, theperiosteum exhibits regenerativepotential; likewise, periosteal graftshave been used to treat bone fractures

Conflict of interest and source of

funding statement

The authors declare that they have noconflict of interest. This research pro-ject was supported by a grant from theCalifornia Institute of RegenerativeMedicine (CIRM) TR1-01249.

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd1170

J Clin Periodontol 2014; 41: 1170–1177 doi: 10.1111/jcpe.12310

and cartilage injuries (Baums et al.2007, Giannoudis et al. 2011, Liet al. 2012, Soldado et al. 2012).Periosteal grafts are not commonlyemployed in oral surgical proce-dures, and in periodontology, it isthought that osteogenic cells arederived from undifferentiated peri-vascular connective tissue cells orpericytes (Davies 2003). In fact, mostassumptions about bone healing areextrapolated from experimentalmodels of skeletal repair in longbones, like that of Duhamel’s earlyperiosteal studies.

Because of their histological simi-larities and equivalent mineral densi-ties, most investigators presupposethat the periosteum of long bonesand of craniofacial bones is analo-gous. A number of unique features,however, distinguish the appendicu-lar skeleton and its associated peri-osteum from the craniofacialskeleton and its periosteum. Forexample, craniofacial and appendic-ular periostea are differentiallyaffected by anabolic agents such asbisphosphonates (Adamo et al.2008, Reid 2009, Silverman & Lan-desberg 2009, Knight et al. 2010).Leucht et al. (2008) used a lineagelabelling strategy to demonstratethat craniofacial and long bone peri-ostea contribute differently to bonerepair: in long bones, periostealstem/progenitor cells are derivedfrom mesoderm, whereas in cranio-facial bones, the periosteal stem/pro-genitor populations are derivedfrom the neural crests (Leucht et al.2008).

Here, we sought to expand ourunderstanding of the differencesbetween craniofacial and long boneperiosteum. We investigated thecharacteristics and regenerativecapacity of the maxillary perios-teum, using the long bone perios-teum as a control. Bone loss,especially vertical bone loss, in eden-tulous ridges of partially dentatepatients constitutes one of the majorsurgical challenges in oral surgery(McAllister & Gaffaney 2003, Roc-chietta et al. 2008, Esposito et al.2009), so a deeper understanding ofthe maxillary periosteum and itsmanagement in surgery could allowus to harness its regenerativepotential for improvement of osseo-integration and vertical bone regen-eration in oral surgery.

Materials and Methods

Animal care

All procedures followed protocolsapproved by the Stanford Commit-tee on Animal Research. Animalswere housed in a temperature-con-trolled environment with 12-h light/dark cycles and were given soft dietfood (Bio Serv product #S3472) andwater ad libitum. No antibioticswere given to the animals and therewas no evidence of infection or pro-longed inflammation at the surgicalsite.

Periosteal surgery

Adult wild-type mice (males,between 3 and 5 months old) wereanaesthetized with an intraperitonealinjection of ketamine (80 mg/kg) andxylazine (16 mg/kg). The mouth wasrinsed using a povidone-iodine solu-tion for 1 min.

In cases where the tunnel tech-nique was used, a small incision wasmade behind the maxillary incisor,perpendicular to the crest. Subse-quently, a periosteal was gentlyinserted into the incision, with bonecontact, raising the periosteum infull thickness along the crest, towardthe first molar (in tunnel technique)(N = 17). An analogous injury wasmade in the tibia, with a small inci-sion made on the proximal tibia,perpendicular to the crest, followedby insertion of a periosteal, raisingthe periosteum distally, in full thick-ness along the medial surface(N = 15). In cases to evaluate thepotential for vertical maxillary boneregeneration, an absorbable collagensponge (HELISTAT, ref.1690-ZZIntegra LifeSciences Corporation)was inserted into the tunnel (N = 5).

The surgical site was carefullyrinsed with 0.9% sodium chloride,and the wound was sutured closedwith non-absorbable single inter-rupted sutures (Ethilon monofila-ment 9-0, Johnson & JohnsonMedical, New Brunswick, NewJersey, USA). Following surgery,clinical examinations were performedand the mice received subcutaneousinjections of buprenorphine (0.05–0.1 mg/kg) for analgesia each dayfor 3 days. Surgeries on the left sidesof the mice were performed 1 weekafter the right sides to obtain two

different time points per mouse.Mice were sacrificed in order toobtain each of the following timepoints: 1, 3, 7, 14, 21, and 28 dayspost-surgery.

Sample preparation, processing, and

histology

Maxillae and tibiae were harvested,the skin and outer layers of musclewere removed, and tissues werefixed in 4% paraformaldehyde over-night at 4°C. The samples weredecalcified in a heat-controlledmicrowave in 19% EDTA for2 weeks. After demineralization,specimens were dehydrated throughan ascending ethanol series prior toparaffin embedding. Eight-micron-thick longitudinal sections were cutand collected on Superfrost-plusslides for histology includingMovat’s pentachrome, Aniline blue,H&E, Safranin-O, and Picro siriusred staining.

Cellular assays

Alkaline phosphatase (ALP) activitywas detected by incubation in nitroblue tetrazolium chloride (NBT;Roche, Indianapolis, Indiana, USA),5-bromo-4-chloro-3-indolyl phos-phate (BCIP; Roche), and NTMbuffer (100 mM NaCl, 100 mM Tris,pH 9.5, 5 mM MgCl). Tartrate-resis-tant acid phosphatase (TRAP) activ-ity was observed using a leukocyteacid phosphatase staining kit(Sigma). After developing, the slideswere dehydrated in a series of etha-nol and xylene and subsequentlycover-slipped with Permount mount-ing media.

For terminal deoxynucleotidyltransferase dUTP nick end labelling(TUNEL) staining, sections wereincubated in proteinase K buffer(20 µg/ml in 10 mM Tris, pH 7.5),applied to a TUNEL reaction mix-ture (In Situ Cell Death DetectionKit, Roche), and mounted withDAPI mounting medium (VectorLaboratories). Slides were viewedunder an epifluorescence micro-scope.

Immunohistochemistry

Tissue sections were deparaffinizedfollowing standard procedures.Endogenous peroxidase activity was

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Regeneration via maxillary periosteum 1171

quenched by 3% hydrogen peroxidefor 5 min, and then slides werewashed in PBS. Slides were thenimmunostained using a proliferatingcell nuclear antigen (PCNA) stainingkit (Invitrogen, Frederick, MD,USA, Ref: 93-1143), following man-ufacturer’s protocol.

Results

Periostea differ by anatomic location

Although maxillary and mandibularvertical bone loss is a major issue inageing patients with missing teeth(Cawood & Howell 1988), periosteaof these craniofacial bones have lar-gely escaped analysis. As the perios-teum is a source of stem cells(Malizos & Papatheodorou 2005),we focused our analyses on the peri-ostea associated with these bones,and compared these results withanalogous data from the tibia.

By histological analyses, the firstobvious distinction between the max-illa and tibia was the thickness ofthe periosteum: the tibial periosteumwas thick and the ratio of cells/stroma was high (Fig. 1a). In con-trast, the oral maxillary periosteumwas thin and the cells/stroma ratiowas considerably lower (Fig. 1b).Clinicians appreciate that the tibialperiosteum is loosely adherent to thesurrounding muscle tissue, whichallows for easier movement of theperiosteum, while the maxillary peri-osteum is tightly bound to the sur-rounding connective tissue,restricting movement (Shaly Bothla:Periodontics Revisited 2011). Cellproliferation was another distinctionbetween the periosteal. Cellsimmuno-positive for proliferatingcell nuclear antigen (PCNA) werefound throughout the tibia perios-teum (Fig. 1g), while the maxillaryperiosteum was devoid of PCNA+ve

cells (Fig. 1h). Nearby gingival epi-thelial cells were PCNA+ve, showingthat the immunostaining was suc-cessful (not shown). Collectively,these data indicated the intact tibialperiosteum was more proliferativethan its maxillary counterpart,resulting in a thicker, more denselycellular tissue.

We evaluated alkaline phospha-tase (ALP) activity as a measure ofmineralization (Gerstenfeld et al.1987) and found in the tibia that

activity was high in the periosteumand low in the endosteum (Fig. 1i).In the maxilla, ALP activity was

high on the oral surface low on thenasal surface of the periosteum(Fig. 1j). Using tartrate-resistantacid phosphatase (TRAP) stainingto detect osteoclast activity (Minkin1982), we found that in the tibia,TRAP activity was limited to theperiosteum and was undetectable inthe endosteum (Fig. 1k). In themaxilla, TRAP activity was limitedto the nasal periosteum, with nodetectable activity in the oral perios-teum (Fig. 1l). These observationssuggest that in the intact bone, min-eralization predominates on the oralsurface of the intact maxilla,whereas resorption predominates onthe nasal surface. This is in agree-ment with published reports (Ather-ton et al. 1974). Collectively, thesedata indicate that the morphologyand cellular activity of the tibialand maxillary periostea are mark-edly different. This conclusion sup-ports the widely establishedconclusion that periostea differ byanatomical location (Chang &Knothe Tate 2012).

A surgical model of mechanical

stimulation to the periosteum

Post-development, the bone-formingpotential of the periosteum is reacti-vated by trauma, infection, and insome cases, growing tumours (Mali-zos & Papatheodorou 2005). Toevaluate the bone-forming potentialof the maxillary periosteum, wedeveloped an animal model inwhich a standard, minimally inva-sive technique was used to elevatethe periosteum away from theunderlying bone. The general orga-nization of the maxillary periosteumrelative to alveolar bone, its overly-ing connective tissue and gingiva, aswell as the olfactory epitheliumwere determined (Fig. 2a). The sur-gical approach began with an inci-sion down to the alveolar bonefollowed by elevation of the tissuewith a blunt periosteal elevator(Neubauer et al. 2011, Masic et al.2012), resulting in a tunnel betweenthe connective tissue/periosteum andthe periosteum/bone surface(Fig. 2b–e).

We evaluated periosteal and con-nective tissues immediately after thetunnelling procedure. We noted thestabilization of a clot within the tun-nel (dotted lines), with little evidence

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Fig. 1. A histological and cellular com-parison of tibial and maxillary periostea.Histological staining of representativelongitudinal tissue sections through thetibia and the maxillary crest (a–b0) penta-chrome staining, (c, d) H&E, (e, f) Safra-nin-O (Saf-O). Proliferating cell nuclearantigen (PCNA) immunostaining is pres-ent in (g) the tibial periosteum and (h) isundetectable in the maxillary periosteum.Alkaline phosphatase (ALP) activity isdetectable in osteoblasts of (i) the perio-steal surface of the tibia and (j) the oralsurface of the maxillary crest. Tartrate-resistance acid phosphatase (TRAP)staining is detectable in osteoclasts (k)periosteal surface of the tibia and (l) lin-ing the nasal surface of the maxillarycrest. Scale bars: (a–l) 50 lm. Abbrevia-tions: eo, endosteum; cb, cortical bone;po, periosteum; oe, oral epithelium; mc,maxillary crest; ct, connective tissue; g,gingiva; op, oral periosteum; np, nasalperiosteum.


1172 Mouraret et al.

of inflammation (Fig. 2f). On post-surgery day 14, a well-organized, den-sely cellular fibrous tissue occupied

the tunnel (dotted lines, Fig. 2g). Theconnective tissue immediately abovethe tunnel appeared normal, without

evidence of an inflammatory infiltrate(Fig. 2g). By post-surgery day 28,cells within the tunnel were furtherorganized and areas of bone resorp-tion under the tunnel were observed(arrows, Fig. 2h).

An analogous procedure was per-formed in the tibia, with a bonedepth incision followed by periostealelevation that resulted in a periostealtunnel (Fig. 2i,k). On post-surgeryday 1, histological analyses showed adensely cellular response associatedwith slight periosteal thickening(Fig. 2l). By post-surgical day 14,further periosteal thickeningoccurred (Fig. 2m), and by day 28,areas of new woven bone wereobserved (Fig. 2n). Thus, the sameinjury performed in maxilla and intibia resulted in radically differentresponses. In the maxilla, boneresorption ultimately occurred inresponse to the tunnelling procedurewhile in tibia, the response to tunnel-ling was bone formation.

Relative to the tibia, the maxillary

periosteum exhibits little bone-forming

potential

At post-surgical day 3, histologicalanalyses revealed robust and denselycellular soft tissue response to perio-steal elevation in tibia and a minimalcellular response tunnel in maxilla(Fig. 3a–f). PCNA staining showeda strong proliferative response in thetibial periosteum (Fig. 3g), but aminimal response in the maxillaryperiosteum (Fig. 3h). Minimal celldeath was detected in response tothe tunnelling procedure in the tibia(Fig. 3i), whereas marked cell deathwas evident in the maxilla (Fig. 3j).Analyses of osteoclast activityrevealed that TRAP staining was vir-tually undetectable in the tibia(Fig. 3k). In contrast, TRAP+ve cellswere evident throughout the maxil-lary periosteum (Fig. 3l). Theseobservations suggest that the tibiadisplayed a stronger repair responseto the tunnelling procedure thanthose observed in the maxillary peri-osteum.

At post-surgical day 7, mineral-ized tissue had formed at the site ofthe tunnelling procedure in the tibia(Fig. 3m,m0; see also Fig. 3o,q). Theresponse in the maxillary periosteumremained unremarkable (Fig. 3n,n0;see also Fig. 3p,r). PCNA staining

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Fig. 2. Periosteal tunnelling technique. (a) Pentachrome staining of a representativelongitudinal tissue section through the maxillary crest, pre-operatively. (b) Schematicillustration of the incision site and elevation of a full-thickness periosteal flap using atunnelling procedure. Intraoperative photographs of the maxillary tunnelling proce-dure include (c) the incision site, (d) the tunnelling and elevation, and (e) the site ofsuturing. Pentachrome staining of representative longitudinal tissue sections throughthe maxillary crest on (f) post-surgical day (PSD) 1 illustrating the tunnel filled with ablood clot (dotted line, and inset); note the preserved architecture of the surroundingconnective tissues. (g) On PSD14, illustrating the tunnel occupied by a population ofdense, flattened cells (dotted line, and inset) and loosely organized connective tissue.(h) On PSD28, the neo-periosteum covers the partially resorbed oral surface of themaxillary crest (dotted line and inset). Analogously, intraoperative photographs of thetibial tunnelling procedure include (i) the incision site, (j) the tunnelling and elevation,and (k) the site of suturing. Pentachrome staining of representative longitudinal tissuesections through the tibia on (l) PSD1. (m) On PSD14. (n) On PSD28. Scale bars: (a,f–h, l, m) 50 lm. Abbreviations: oe, olfactory epithelium; mc, maxillary crest; ct, con-nective tissue; g, gingiva; cb, cortical bone; po, periosteum; bm, bone marrow.



showed continued proliferation intibial periosteum (Fig. 3s); there wasno detectable proliferation in maxil-lary periosteum (Fig. 3t). ALP stain-ing was robust in the tibia (Fig. 3u),but remained at baseline in maxilla(Fig. 3v). TRAP staining for osteo-clast activity was notable in both tib-ial (Fig. 3w) and maxillary (Fig. 3x)responses.

The maxillary periosteum has a delayed

cellular response to the tunnelling

procedure

At post-surgery day 21, histologicalanalyses showed new woven boneformation in tibia (Fig. 4a,a0; seealso Fig. 4c,e) and bone resorption(resorptive area; RA) in maxilla(Fig. 4b,b0; see also Fig. 4d,f).PCNA staining was still detectable

in the tibia (Fig. 4g). Proliferationremained virtually undetectable inthe maxilla (Fig. 4h). Overall, ALPactivity was diminished compared toearlier time points (Fig. 4i), suggest-ing that the mineralization phasewas largely complete in the tibia.For the first time since the injury,

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Fig. 3. Early tibial and maxillary response to injury. At post-surgical day (PSD) 3, his-tological analyses of representative longitudinal tibial and maxillary tissue sectionsstained with (a–b0) pentachrome, (c, d) H&E, and (e, f) Saf-O. PCNA immunostainingshowing proliferating cells in (g) the tibia and (h) the maxilla. Terminal deoxynucleot-idyl transferase dUTP nick end labelling (TUNEL) staining of (i) tibial and (j) maxil-lary response showing apoptotic cells in green and nuclei in blue. TRAP staining of(k) tibial and (l) maxillary response. At PSD7, histological analyses of tibial and max-illary responses stained with (m–n0) pentachrome, (o, p) H&E, and (q, r) Saf-O.PCNA staining of (s) proliferating cells in the tibia and (t) maxilla. ALP staining ofosteoblast activity (u) in the tibia and (v) in the maxilla. TRAP staining in both the(w) tibia and (x) maxilla. Scale bars: (a–x) 50 lm. Abbreviations: cb, cortical bone;po, periosteum; mc, maxillary crest; ct, connective tissue.

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Fig. 4. Periosteal tunnelling inducesosteogenesis in the tibia and bone resorp-tion in the maxilla. At PSD 21, histologi-cal analyses of representativelongitudinal tissue sections through theinjured tibia and maxillary crest, stainedwith (a–b0) pentachrome, (c, d) H&E,and (e, f) Saf-O. PCNA staining of (g)the tibial response, and (h) the maxillaryresponse. ALP activity in (i) the tibialresponse and (j) the maxillary response.TRAP staining of (k) the tibial responseand (l) the maxillary response. Scalebars: (a–l) 50 lm. Abbreviations: cb, cor-tical bone; po, periosteum; mc, maxillarycrest; ct, connective tissue; t, tunnel; RA,resorptive area.



strong ALP activity was evident inmaxilla (Fig. 4j). Bone remodellingwas largely complete in the tibia, asindicated by minimal TRAP staining(Fig. 4k). In contrast, TRAP stain-ing of osteoclast activity was robustin the maxilla (Fig. 4l). Results fromthese late-stage analyses suggest thatrelative to the tibia, the maxillaryperiosteum has a delayed andresorptive response to the tunnellingprocedure.

Insertion of collagen sponge into the

periosteal tunnel induces vertical bone

regeneration in the maxilla

In order to investigate the potentialfor the maxillary periosteum toregenerate bone, we inserted a colla-gen sponge into the periosteal tunnel(Fig. 5a), effectively creating spacein which bone could form. Indeed,in clinical practice, achieving verticalbone regeneration is extremely diffi-cult due to the collapse of availablespace for the new osteoid tissue. Byinserting a collagen sponge into theperiosteal tunnel, we provided thescaffolding necessary to maintain the

space in which new bone couldform.

By 28 days after the procedure,we noted an area of robust newbone formation (black dashed line,Fig. 5b). We analysed two areas ofinterest: the area near the collagensponge (red line, Fig. 5b,c–h) andthe site of bone regeneration (blackline, Fig. 5b,i–n). Histological analy-ses revealed an area where the colla-gen sponge remained without newbone formation (Fig. 5c–e), andDAPI nuclear staining showed littlecellular infiltrate into this area(Fig. 5f). Within the collagensponge, ALP activity and TRAPstaining were restricted to the alveo-lar bone surface (Fig. 5g,h), similarto the analogous intact counterpart.Histological analyses revealed anarea of new, woven bony regenerate(Fig. 5i–k). Picro sirius red stainingof the bony regenerate revealed ori-entation of collagen fibres parallel tothe tension vector caused by thetenting of the periosteum with thecollagen sponge (Fig. 5l). Within thebone regeneration site, high ALPactivity and TRAP staining were

observed in the newly formed matrix(Fig. 5 m and n), indicating healthybone turnover throughout the verti-cally regenerated bone. Immunohis-tochemical analyses of proliferationin the two areas revealed little to noproliferation in both areas (data notshown), suggesting that cells in thenew bone area likely migrate intothe collagen sponge, colonizing andmineralizing the scaffold. In addi-tion, the major proliferation burstlikely occurred before post-surgeryday 28.

Discussion

Periosteal characteristics differ with

anatomical location

The physical and cellular characteris-tics of periostea differ with anatomi-cal location (Leucht et al. 2008). Inclinical practice, the tibial perios-teum is loosely attached to overlyingmuscle, while the maxillary perios-teum is tightly adherent to overlyingconnective tissue (Popowics et al.2002). In the intact mouse tibia, weobserve that much of the endoge-

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Fig. 5. Vertical maxillary bone regeneration occurs with insertion of collagen sponge into maxillary periosteal tunnel. (a) Schematicof injury model with collagen sponge inserted into periosteal tunnel, between elevated periosteum and maxillary crest. Representa-tive longitudinal tissue sections through the injured maxillary crest on PSD28, stained with (b) pentachrome showing un-resorbedcollagen sponge (red dotted line) and new bone (black dotted line). The un-resorbed collagen sponge stained with (c) pentachrome,(d) H&E, (e) aniline blue, and (f) DAPI. Both (g) ALP and (h) TRAP staining restricted to the surface of the maxillary crest. Thenew bony regenerate stained with (i) pentachrome, (j) H&E, and (k) aniline blue, all showing areas of newly deposited woven bone.(l) Picro sirius red staining shows orientation of new collagen fibres parallel to direction of periosteal tenting. Both (m) ALP and(n) TRAP staining is robust within the area of new woven bone. Scale bars: (b–n) 50 lm. Abbreviations: cs, collagen sponge; nb,new bone; mc, maxillary crest.



nous bone remodelling activity isfound on the periosteal surface(Fig. 1 and see Leucht et al. 2007),whereas in the intact mouse maxilla,bone deposition occurs along theoral periosteum and bone resorptionalong the nasal periosteum (Fig. 1).This physical separation betweensites of bone deposition and boneresorption suggests that the size ofthe sinus changes with growth (Ath-erton et al. 1974).

Molecular, cellular, and geneticstudies demonstrate that long boneperiostea are more osteogenic thanflat bone periostea (Chang & KnotheTate 2012), but that craniofacialperiostea enclose cells with greatermulti-potency (Leucht et al. 2008).These apparent differences highlightthe need for an in-depth understand-ing of the bone-forming potential oforal periostea, which has direct rele-vance for bone reconstructive proce-dures in oral surgery. Stimulation oflong bone periostea induces an oste-ogenic, regenerative response (Gold-man & Smukler 1978), but here weshow that the maxillary periosteumhas a delayed, attenuated, or oppo-site response to the same injury. Forexample, the periosteal tunnellingprocedure can induce robust boneformation in the tibia while the verysame procedure can induce boneresorption in the maxilla (Figs 3 and4).

Maxillary periosteal elevationalone did not result in vertical boneregeneration (Fig. 4). We used a col-lagen sponge to provide scaffoldingand to maintain space, as both func-tions are essential for successful tis-sue regeneration (Sturm et al. 2010,Chen et al. 2011). When the perio-steal tunnel architecture is main-tained and the periosteum tentedaway from the maxillary crest with acollagen sponge, bone formation canoccur (Fig. 5). The highly specificorganization of the new collagenfibres parallel to the direction of theperiosteal tenting suggests that theintroduction of a collagen spongeprovided the necessary mechanicalenvironment for bone regenerationto occur. There is, however, acaveat: despite the fact that success-ful bone regeneration was observedin this model system, it is importantto note that mice regenerate muchfaster than humans (Auer et al.2007). In addition, the experiments

shown here were performed in younganimals. In humans, it is likely thata material such as the collagensponge would resorb much fasterthan the human tissue would regen-erate, and thus the mechanical sta-bility provided by this scaffoldwould be lost before the pro-osteo-genic periosteal tissue would occupythe site.

Collectively, our data here sug-gest that regenerative techniques thatutilize long bone periosteum cannotsimply be extrapolated to craniofa-cial applications. Further study ofthe anatomically specific cellular andmolecular characteristics of variousperiostea is needed in order to har-ness the full regenerative capacity ofthe oral periosteum.

Acknowledgements

Special thanks to Dr. JingTao Li(Stanford University, Division ofPlastic and Reconstructive Surgeryand West China Stomatology Hospi-tal, China) for contributions regard-ing clinical implications andsignificance.

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Address:Jill HelmsRoom GK 207257 Campus DrStanford, CA 94305USAE-mail: [email protected]

Clinical Relevance

Scientific rationale for the study: Inthe ageing population, there is aclinical need for vertical alveolarbone regeneration. As a source ofosteogenic cells, the maxillary peri-osteum might be a useful tool toaddress this need.

Principal findings: Using periostealtunnelling procedures in maxilla andtibia, it was demonstrated that longbone and craniofacial periostearespond differently to injury andthat, in the appropriate mechanicalenvironment, maxillary periosteum

can induce vertical alveolar boneregeneration.Practical implications: Resultssuggest a potentially useful methodfor surgical management andmanipulation of the maxillaryperiosteum to achieve vertical boneregeneration.



the potential for vertical bone regeneration via maxillary...

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