HUMAN MANDIBLE BONE DEFECT REPAIR BY THE GRAFTING OF DENTAL.pdf

75www.ecmjournal.orgR dAquino et al.DPCs repair human mandible defectsEuropean Cells and Materials Vol.18 2009(pages 75-83)ISSN 1473-2262AbstractIn this study we used a biocomplex constructed from dentalpulp stem/progenitor cells(DPCs)and a collagen spongescaffold for oro-maxillo-facial(OMF)bone tissue repairin patients requiring extraction of their third molars.Theexperiments were carried out according to our InternalEthical Committee Guidelines and written informed consentwas obtained from the patients.The patients presented withbilateral bone reabsorption of the alveolar ridge distal tothe second molar secondary to impaction of the third molaron the cortical alveolar lamina,producing a defect withoutwalls,of at least 1.5 cm in height.This clinical conditiondoes not permit spontaneous bone repair after extractionof the third molar,and eventually leads to loss also of theadjacent second molar.Maxillary third molars wereextracted first for DPC isolation and expansion.The cellswere then seeded onto a collagen sponge scaffold and theobtained biocomplex was used to fill in the injury site leftby extraction of the mandibular third molars.Three monthsafter autologous DPC grafting,alveolar bone of patientshad optimal vertical repair and complete restoration ofperiodontal tissue back to the second molars,as assessedby clinical probing and X-rays.Histological observationsclearly demonstrated the complete regeneration of bone atthe injury site.Optimal bone regeneration was evident oneyear after grafting.This clinical study demonstrates that aDPC/collagen sponge biocomplex can completely restorehuman mandible bone defects and indicates that this cellpopulation could be used for the repair and/or regenerationof tissues and organs.Keywords:Dental pulp stem/progenitor cells(DPCs),bone,human mandible,stem/progenitor cell graft,bioscaffold,regenerative medicine,clinical study.Address for correspondence:Gianpaolo PapaccioDepartment of Experimental MedicineSection of Histology and Embryology,TERM Division2nd University of Naples,via L.Armanni,5,80138 Naples(Italy)Telephone Number:+39 081-5666014FAX Number:+39 081-5666015Skype:StemfaceE-mail:gianpaolo.papacciounina2.itIntroductionThe aim of tissue engineering(TE)is the regeneration oftissues through the combined use of biomaterials andbiologic mediators in order to provide new tools forregenerative medicine(RM).Over the last years,TE hasmade significant progress,moving from being merely abiomaterial science towards being a genuinelymultidisciplinary field,through the integration of biology,medicine and various engineering sciences.Importantly,future procedures will make increasing use of autologoustransplants(i.e.,material obtained from the sameindividual to whom they will be reimplanted);thus,theneed for immunotherapy will be avoided.Ideally,thesetransplants will possess predictable patterns ofvascularisation and nerve supply,which are both importantaspects for a return to optimal functionality.The need to develop tissue replacement andimplementation strategies is particularly felt in the oro-maxillo-facial(OMF)field.Replacement of OMFstructures is tricky and peculiar because orofacialfunctions such as facial expression,articulation ofspeech,chewing and swallowing are exquisitely delicate,being based on complex three-dimensional anatomicalstructures formed from soft(skin,mucosa and muscle)and hard(craniofacial skeleton and teeth)tissues(Bluteauet al.,2008).The repair and regeneration of bone is a major issuein the OMF field and for the whole human body in general.Bone loss is caused by many diseases(congenital ordegenerative),traumas and surgical procedures;it is aproblem for functionality and is having an ever-increasingsocial impact,especially in elderly subjects.Bone is formed by extracellular matrix(ECM)rich incollagen and elastic fibres adherent to hydroxyapatitecrystals.Adult bone is continuously remodelled throughspecific osteoblast/osteoclast interaction.Stem/progenitorcells residing in the periosteum and endosteum of bonepossess a limited regenerative potential(Salgado et al.,2006).For this reason,surgical intervention usingbiocompatible fillers or bone-grafting techniques isindispensable when significant bone loss occurs.To avoidside effects produced by the use of biocompatiblematerials and/or bone withdrawal,new biotechnologicalapproaches for repair must be envisaged.Although stem/progenitor cells have been isolatedfrom different tissues and extensively studied in vitro andin vivo in the past years,there is no information yet on theapplication of human stem/progenitor cells for the repairof OMF structures at a clinical level.Unfortunately,thereHUMAN MANDIBLE BONE DEFECT REPAIR BY THE GRAFTING OF DENTALPULP STEM/PROGENITOR CELLS AND COLLAGEN SPONGE BIOCOMPLEXESRiccardo dAquino1,2,Alfredo De Rosa1,Vladimiro Lanza1,Virginia Tirino2,Luigi Laino1,Antonio Graziano1,Vincenzo Desiderio2,Gregorio Laino1 and Gianpaolo Papaccio2*1Dipartimento di Discipline Odontostomatologiche,Ortodontiche e Chirurgiche,2Dipartimento di MedicinaSperimentale,Sezione di Istologia ed Embriologia,Tissue Engineering and Regenerative Medicine(TERM)Division,Secondo Ateneo di Napoli,Naples,Italy76www.ecmjournal.orgR dAquino et al.DPCs repair human mandible defectsare limits on the use of stem/progenitor cells in therapy,such as the low number of stem/progenitor cells that canbe collected,morbidity at the site of collection and thedifficulties in reaching the site of repair.Dental pulp is a niche housing neural-crest-derivedstem cells.This niche is easily accessible and there islimited morbidity after collection(Jo et al.,2007;Lenschet al.,2006;Mitsiadis et al.,2007).Previous studies haveshown that dental pulp stem cells(DPSCs)are capable ofdifferentiating into osteoblasts(Laino et al.,2005;Lainoet al.,2006b)that secrete abundant extracellular matrix(ECM)and that can build a woven bone in vitro(Laino etal.,2006a).Furthermore,DPSCs are capable of forming acomplete and well-vascularised lamellar bone after graftinginto immunosuppressed rats(dAquino et al.,2007;Graziano et al.,2008).The quality and quantity ofregenerated bone formed by DPSCs was demonstrated inin vitro and in vivo experiments using stem cells andbiomaterials(dAquino et al.,2008;dAquino et al.,2007;Graziano et al.,2008;Laino et al.,2005).Thus,dentalpulp could be considered as an interesting and potentiallyimportant source of autologous stem/progenitor cells thatare ready for use for therapeutic purposes,such as therepair/regeneration of craniofacial bones.The aim of this study,therefore,was to demonstratethat DPCs could be used to repair bone defects in humans.Here we give evidence that DPCs seeded onto collagensponge bioscaffolds repair alveolar defects of the mandibleproduced after extraction of impacted third molars.Theautografts produced a fast regeneration of bone,which wasof optimal quality and quantity when compared to standardtechniques commonly used for guided bone regenerationand bone grafts of various origins(Jensen et al.,2004).Materials and MethodsEthicsAll procedures described here comply with Internal EthicalCommittee guidelines,approved on June 12th,2005(Second University of Naples Internal Registry:Experimentation#914-Bone repair using stem cells).Patients were invited,before being enrolled for thestudy,to carefully read and sign an informed consent form,drafted by us following instructions from our internalEthical Committee.ObjectivesThe objective of this clinical study was to repair an alveolarbone defect secondary to routine wisdom tooth extraction.Usually,after extraction of impacted unerupted or partiallyerupted wisdom(third molar)teeth,a proportion of patientsrisk reabsorption of the alveolar ridge distal to the secondmolar roots.In these patients,destruction of the toothsocket produces a pocket formed from 2 walls,which arerepresented by the root of the second molar and the distalridge,with the lingual wall forming a third wall whenpresent.This clinical condition(post-extractive alveolarbone loss),in which vertical loss has a probing depth of atleast 7 mm,jeopardizes the second molar in an average offive years or less and does not allow bone repair withnormal techniques(Dodson,2007).Thus,it represents acomplication associated with the removal of the thirdmolars that should not be underestimated.For this reason,we identified patients at risk of post-extractive alveolarbone loss as candidates that could benefit for a boneregeneration therapy.Participants:patient selection and preparationEligibility criteria for participants and settings were thefollowing:extraction needed for all wisdom teeth,withclosely comparable conditions for the two lower impactedteeth;no systemic disease;no pregnancy(for females);noroutine drug use.Patients with two similar lower molarswere needed for the study so that we could use one as atest(T)site and the other one as a control(C)site.Seventeen out of the 100 patients initially contacted forthis study consented to surgery.Of these 17 patients,7(6females and 1 male)returned for the one-year follow-up.The template for the enrolment of patients was set withinthe limits of the approved clinical trial.All the procedureswere performed at the Department of Odontostomatologyof the Second University of Naples.Patients were subjected to professional oral hygieneone week before surgery.They were then instructed toperform domiciliary hygiene of the oral cavity correctly,which consisted in washing the mouth with 0.2%Chlorhexidin(CHX)after tooth brushing,twice a day untilsurgery was performed.Pre-surgery evaluation of dental pulp stem/progenitor cellsBefore embarking on regeneration surgery,we needed toobtain stem/progenitor cells from the pulps of the patients.Patients were therefore subjected to the extraction of theupper(maxillary)molars and the pulps harvested aspreviously described(dAquino et al.,2007).Briefly,teethwere washed in 0.2%CHX solution,the pulp chamberopened using a surgical drill and the pulp collected.Then,the pulp was rinsed in 1.5 ml saline solution andmechanically dissociated;using previously describedprocedures(Graziano et al.,2008).After dissociation,cellswere filtered through a 70m strainer and cultured in-minimal essential medium(MEM)(Cambrex,Charles City,IA,USA)with 20%FBS(Invitrogen,San GiulianoMilanese,Italy)and the medium changed twice a week.At day 21 cells were detached and analysed at theFluorescence Activated Cell Sorter(FACS Vantage,Beckton Dickinson,Franklin Lakes,NJ,USA)for stem/progenitor antigen expression in good manufacturingpractice(GMP)conditions.Cells were detached using0.02%EDTA solution,centrifuged and incubated with 1lof antibody in 100 l of phosphate buffered saline(PBS)solution for 1h at 4C.Antibodies were:anti-CD34(cloneAC136,Miltenyi Biotech,Calderara sul Reno,Bologna,Italy)and anti-flk-1(c.sc-57135,Santa Cruz,CA,USA).Extraction of wisdom teethPatients were prepared for surgery by decontamination ofthe oral cavity with CHX.Then lower(mandible)impactedthird molars were extracted following a standardprocedure:after making a horizontal incision in the gum,77www.ecmjournal.orgR dAquino et al.DPCs repair human mandible defectsthe muco-periosteal flap is reflected and the bone coveringthe tooth is removed using a round bur.The area is irrigatedwith a steady stream of saline solution until the crown isentirely exposed.When the oral surgeon is not able to extract the wholetooth in one go,a groove is created vertically(along thelong axis of the tooth)at the cervical line of the tooth,using a fissure bur,in order to separate the crown from theroot.The groove created by the bur must not be deep,sincethe mandibular canal is often found in close proximity tothe tooth and the risk of injuring or severing the inferioralveolar nerve must be avoided.After being placed in thegroove,a straight elevator is used to separate the crownfrom the root,with a rotary movement.The crown isremoved separately,using the same elevator,with a rotarymovement upwards,and the root is then easily removed,using a straight or angled elevator,the blade end of whichis placed in a purchase point created on the buccal side ofthe root.In the case of a tooth with multiple roots,the crownmust be sectioned and removed,as above described.Afterwards,if the roots of the impacted tooth are separatedduring crown sectioning,they can be easily removed onein succession,starting with the distal root and then themesial root.After smoothing the bone,the area is irrigated withsaline solution and the distal root of the second molar isplanned with a Gracey curette,and all the necrotic tissueis taken away.Stem/progenitor cells,obtained as above described,were gently endorsed with a syringe onto a collagen spongescaffold(Gingistat,Vebas,San Giuliano Milanese,Italy).The sponge-cell implant was used to fill the space left bythe extraction procedure(test(T)site).A sponge withoutcells was used to fill the control(C)site.A flap of gum was then sutured as a tendon in order toavoid any contact with the oral cavity.A suture was thenplaced at the distal portion of the second molar and theothers were placed at the interdental papillae and at theposterior end of the incision.For both sites,a replacementjig was placed to ensure correct localization for samplewithdrawal.Post-surgery patient evaluationsClinical and radiological controls were performed.The firstcontrol was scheduled at day 7 after surgery,when X-ray(for each patient the Ethical Committee permitted toperform a maximum of 4 X-ray Orthopantomographies(OPTs)per year and a maximum of 8 endo-oral X-raysper year),clinical observation and suture removal wereperformed.Oedema,presence of inflammation andfunctionality were clinically evaluated.Patients were controlled once a month thereafter,upto the third month.During these controls,clinicalobservations and X-rays were performed.During thefourth control,at three months after surgery and beforebone sampling,probing depth was performed to evaluatethe retrieved clinical attachment.A sample was then collected from the T and C sites ofeach patient for histological and immunofluorescence(IF)analyses.Each bone sample was collected using a drillwith a replacement jig.Bone specimens were used forhistological observations.For this purpose,each samplewas decalcified in 10%EDTA in distilled water for 2months.Then,each specimen was sectioned and stained.Other than haematoxylin-eosin staining,samples were usedfor IF analyses,using the following antibodies:anti-osteocalcin(OC),anti-osteonectin(ON),anti-bone alkalinephosphatase(BAP),anti-bone morphogenetic protein(BMP)-2 and anti-vascular endothelial growth factor(VEGF)(all purchased from Beckton Dickinson).One yearfrom surgery,further analyses were performed.Statistical analysisStudent t-test(two-tailed)was used for statisticalevaluation.The level of significance was set at p0.05.ResultsThe dental pulp cells of the third maxillary molars collectedfor pre-surgery evaluation were strongly posit