Wound models for periodontaland bone regeneration: the roleof biologic researchANTON SCULEAN, IAIN L. C. CHAPPLE & WILLIAM V. GIANNOBILE

The treatment of periodontal diseases represents asignificant healthcare challenge, and, since the gen-esis of human research endeavours in periodontol-ogy, the ultimate therapeutic goal has been torebuild completely those tissues lost to the diseaseprocess with tissues that are structurally and func-tionally the same (144). Early attempts at regenera-tion were empirical, largely based upon clinicalexperience and included the utilization of scalingand root planing combined with soft-tissue curet-tage or the use of various flap procedures andmaterials for ‘bone grafting’ (17, 30, 94, 100, 101,115, 141). Such approaches occasionally resulted inclinical improvements, expressed as probing depthreductions or bone defect fill, which at the timewere termed ‘new attachment’ or ‘regeneration’ (43,99, 112, 141). However, histologic studies in animalsand humans subsequently demonstrated that suchmethods did not predictably result in the formationof periodontal ligament, root cementum and bone(31, 32, 78, 130).

A proper understanding of the basic biologicmechanisms involved in periodontal wound repairand regeneration requires assessment of the macro-scopic, microscopic, cellular and molecular compo-nents of the healing process (Fig. 1). To determinethe molecular mechanisms that best guide tissueneogenesis during wound repair and regeneration,the outcomes of basic science (laboratory) studiesneeds to be translated to the clinical arena. Thetransfer of discovery research to clinical research,and eventual adoption in clinical practice, requirespre-clinical animal model studies to determine thesafety and early-stage effectiveness of new technolo-gies. This volume of Periodontology 2000 highlightsadvances from a myriad of approaches to determine

comprehensively how wound-healing models areemployed in oral, periodontal and craniofacial tis-sue regeneration. In this introduction we presentthose requirements for the use of pre-clinicalresearch in periodontal wound repair and identifylimitations of the work that would need to beaddressed before human studies in pre-clinicalmodels or directly in the human clinical trial setting(as a result of the limitations of animal models toreplicate the human clinical disease scenario).Finally, the key findings of the reviews within thisvolume of Periodontology 2000 are summarized.

A historical perspective

The history of periodontal regeneration over the pastcentury has recently been summarized (44). In themodern era of regenerative biology, pioneering workwas carried out in the late 1960s by Tony Melcher,who demonstrated, through his studies, advances inthe biology of periodontal wound healing. In 1969,Melcher distinguished the processes or ‘repair’ and‘regeneration’ (82–84) and, in 1976, he postulated that‘the first cells to repopulate a root surface will dictatethe nature and quality of tissue that forms there’ (82).Half a century later, it is perhaps worthwhile reflect-ing upon the extent to which our current treatmentconcepts are biologically based, as Melcher taught, orwhether we still, to some degree at least, fall into thetrap of empiricism when aiming to regenerate peri-odontal defects.

Important questions that arise when exploring bio-logic approaches to periodontal regeneration include:� is there a rationale and a role for pre-clinical (ani-

mal) models to evaluate the biologic potential of

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Periodontology 2000, Vol. 68, 2015, 7–20 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Printed in Singapore. All rights reserved PERIODONTOLOGY 2000

novel regenerative technologies, and what is theclinical relevance of such models?

� are regenerative materials that have been vali-dated using pre-clinical models also effective inthe human situation?

� what are the current concepts in managing angu-lar (intrabony), furcation and recession defectsaround teeth?

� what options are available for managing defectscaused by infection around dental implants?

� what is the role of ‘surgical technique’, includingflap management and suturing, in determiningimprovements in clinical outcomes?

Several decades ago, because of a paucity of knowl-edge regarding the etiology and pathogenesis of peri-odontitis, it was believed that bone was the onlytissue that played a role in supporting the toothwithin the alveolus, and that the ‘diseased bone’ wasprimarily responsible for the presence of angular (in-trabony) or furcation defects arising in periodontitispatients (88, 106). Such beliefs consequently led tothe development of treatment protocols that focussedprimarily on filling the ‘holes’ with a great variety ofbone-grafting materials. The latter were largely non-resorbable and thus effectively acted as inactive bio-logic fillers. Moreover, the majority of studiesevaluating the effects of different surgical procedures

aimed at defect fill with bone grafts and onlyemployed clinical outcome measures, such asprobing pocket depth, probing attachment level,radiological analysis and direct visualization, follow-ing surgical re-entry procedures (21, 43, 52, 87, 99,105, 107, 112). However, such approaches did notfacilitate the determination of true periodontal regen-eration, an outcome that requires systematic histo-logic investigation. Subsequently, pivotal histologicstudies provided evidence that the outcomes of peri-odontal probing were strongly correlated with thelevel of soft-tissue inflammation present and, as such,may not necessarily reflect genuine tissue regenera-tion. For instance, if the gingiva is inflamed, the tip ofthe probe will easily penetrate the pocket basethrough the connective tissues and to a level apical tothe base of the pocket. If inflammation is reduced,the gingival connective tissues contract and demon-strate greater resistance to probing, thus preventingthe tip of the probe from reaching the pocket base(12, 77, 138).

Subsequent research, using different grafting mate-rials in periodontal defects, led to improvements inprobing pocket depths as well as radiographic bonefill, giving the impression that true regeneration hadoccurred. Similarly, hard-tissue infill detected duringclinical re-entry procedures was interpreted as proof

Fig. 1. Phases during periodontal wound repair and regeneration. Increased numbers of fibroblasts from HumanaPress, with permission. From Padial-Molina et al. (95). ECM, extracellular matrix; PDL, periodontal ligament; PMN,polymorphonuclear neutrophil.

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of periodontal regeneration. A greater understandingof the biology of periodontal wound healing andregeneration commenced with the publication of aseries of studies employing pre-clinical models withligature-induced periodontitis, in order to mimicchronic periodontal defects in humans. The resultswere somewhat disappointing and demonstrated thatall treatment procedures aiming to regenerate peri-odontal tissues, including root planing and soft-tissuecurettage, and indeed modified Widman flap surgerywith or without various bone grafts, consistentlyresulted in the formation of a long junctional epithe-lium on the instrumented root surface, with no, orlimited, formation of cementum with inserting peri-odontal ligament fibers (32). Moreover, if bone forma-tion was evident histologically in some of the defects,an epithelial lining was always interposed betweenthe newly formed bone and the root surface. Thesefindings were later confirmed in several independentstudies, demonstrating that the majority ofapproaches to periodontal regeneration remainedempirical in nature and lacked a foundation in bio-logic science (53, 64, 102, 135).

Almost simultaneously with the experiments ofCaton and co-workers were Melcher’s reports from aseries of elegant experiments which had led him tohypothesize that the cells responsible for periodontalregeneration were resident within the periodontal lig-ament and not in the bone itself (82). Thus, Melcherproposed the periodontal ligament as the key tissueresponsible for periodontal regeneration. Thishypothesis was later tested in a series of animal stud-ies, which provided evidence that only the granula-tion tissue originating from the periodontal ligamenthad the capacity to form new cementum and eventu-ally new alveolar bone, and that the connective tissueoriginating from the gingival flap or the alveolar bonedid not promote true periodontal regeneration, asevidenced by the formation of new cementum withinserting collagen fibers and new alveolar bone (55,61, 63, 64, 76, 89–91).

Knowledge obtained from such pre-clinical (animal)models led to the development of the treatment con-cept termed ‘guided tissue regeneration’ (47, 48, 93).The guided tissue regeneration concept built uponMelcher’s thesis that the first cells to populate the rootsurface would dictate the tissue that formed there.Therefore, barrier membranes were developed as bar-riers to the downgrowth of the junctional epithelium,whilst, at the same time, occluding the ingress of fi-broblasts from the gingival connective tissues liningthe surgical flap. Such a dual barrier facilitated thosecells arising from the intact periodontal ligament to

repopulate the debrided root surfaces. This series ofwell-designed and reported experiments (62) is justone example of how a deeper biologic understandingof tissue structure and function may ultimately trans-late into a clinically relevant treatment concept, onceinitially tested in animal models. Furthermore, theselandmark studies highlighted the need for a greaterunderstanding of the biologic processes involved inwound healing before developing novel treatmentconcepts, and the potential dangers of introducingtreatment methods without previously evaluatingthose concepts in pre-clinical models.

Periodontal regeneration: myth orreality?

A plethora of different surgical techniques involvingthe implantation of various types of bone graft and/orbone substitutes, root-surface demineralization proce-dures, guided tissue regeneration, use of growth/dif-ferentiation factors, enamel matrix proteins or variouscombinations thereof, have been employed in order toachieve periodontal regeneration, and have beenshown to promote periodontal regeneration in ani-mals (62, 72, 80, 105, 125, 133, 135). Despite positiveobservations in animal models and successful out-comes reported for many of the available regenerativetechniques and materials in patients, including histo-logic evidence, robust information on the degree towhich reported clinical improvements reflect trueperiodontal regeneration remains limited (19).

When analyzing the literature on human studiesevaluating healing of human intrabony defects fol-lowing the use of various regenerative materials, it isapparent that periodontal regeneration in humanscan be achieved to a variable extent with severalmaterials, including guided tissue regeneration, bio-logic factors, certain types of grafts and numerouscombinations of the aforementioned (127). In con-trast to the findings in animal studies, the implanta-tion of alloplastic materials alone has demonstratedat best limited, and frequently no, periodontal regen-eration in humans (19, 56, 127, 134). These findingsappear to indicate that complete periodontal regener-ation in humans is possible in certain clinical situa-tions and with a limited number of regenerativematerials or their combinations. Whilst studies ofhuman defects may provide important informationon the biologic potential of various regenerative pro-tocols and biomaterials, the information obtainedfrom such studies needs to be interpreted carefully inthe light of the available evidence from pre-clinicaland clinical studies.

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What are the benefits andlimitations of pre-clinical modelsfor research into periodontalregeneration and wound healing?

Wound healing principles need to be carefullyconsidered in the development of biomimetic mate-rials that can provide an appropriate microenviron-ment for tissue formation (72). These materials areused as matrices for the delivery of cells, moleculesor scaffolding constructs for eventual clinical use.Based on these needs, pre-clinical studies haveenabled the initiation of clinical investigations thathave resulted in the approval of new medical devicesor drugs that promote periodontal tissue repair (46).Pre-clinical study plans must be adapted for the spe-cific purpose of the investigation (i.e. mechanistic oroutcomes that will link to a surrogate clinical mea-sure). An example of a mechanistic goal might be todetermine the role of a newly discovered cell-adhe-sion molecule during the induction of experimentalperiodontitis in a genetically modified animal(knockout or transgenic) model. By contrast, a newdrug to stimulate periodontal ligament formationmay be injected into the gingival tissues to examine,histologically, the soft-tissue repair processes at themicroscopic level. A preclinical model is usedbecause this approach is not readily achievable inhumans (at least in a meaningful, adequately pow-ered study). Moreover, the objectives of a pre-clini-cal study must relate to the efficient and effectivedesign of a reconstructive therapy. The choice ofend point is therefore a critical issue in the studydesign. Overall, planning a pre-clinical study to

evaluate regenerative devices requires decisions onanimal species, defect type, the study end point andstudy duration (96). In addition, end points must bedefined to estimate the sample size necessary toachieve the desired power (20). Consideration of thesize of the defects, as well as morphologic changesrelated to the anatomic defect that can occur overtime, can help to estimate the appropriate studyduration.

The selection of pre-clinical models considers thediffering anatomy and healing characteristics ofrodents and larger animals (129). A summary ofpreclinical models commonly used to evaluate peri-odontal-reconstructive therapies, highlighting thoserelated to devices and biologics, is illustrated inTables 1 and 2. Many new regenerative approachesthat strive to demonstrate a proof-of-concept orearly-stage mechanistic understanding of the wound-healing process will employ small animal models asscreening tools before embarking on larger animalstudies (132).

Pre-clinical investigations to promote thedevelopment of new regenerativemedicines

It typically takes over 10 years and more than$US350 million to bring a new drug from the labora-tory to the market (59). It can take 3–4 years of testingin the laboratory and pre-clinical phases before suffi-cient results are available for an application to be sub-mitted to the US Food and Drug Administration,European Medicines Agency or other national/inter-national regulatory authorities to launch clinical inves-tigations (Fig. 2). Only one in 1,000 compounds that

Table 1. Advantages and disadvantages of rat defect models

Defect type Advantages Disadvantages

Fenestration (periodontal) Gives a proof-of-concept in a short intervalWell contained defectsNo gingival tissue ingrowth

Narrow healing windowSmall size, surgical microscopes requiredSpontaneous healing

Capsule (vertical ridge) Standardized shape-dimensionShielded from local environmentFilled solely with cells and fluidsemanating from the residual bone

Not applicable to alveolar boneIsolation from oral environmentSpontaneous regenerationFormation of bone outside the capsule

Alveolar socket Easy and fast to performReproduces well the eventsoccurring in bone healing

Small sizeRapid bone repair comparedwith that for human

Infrabony peri-implantdefect

Surgically createdShort time needed to generate the defectStandard morphology-dimension

Surgically createdSpontaneous regenerationNarrow evaluation window

Reproduced from Pellegrini et al. (96), with permission.

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undergo this initial phase of testing will ever reach thenext phase of testing in humans (33). Assumingregulatory approval for human testing followingsubmission of an Investigational New Drug Applica-tion, three phases of clinical trials typically follow:� Phase I, which establishes safety of a drug.� Phase II, which gains preliminary information rel-

ative to the efficacy of the agent.� Phase III, which is designed to be randomized and

controlled in order to determine the effectivenessof the biologic agent (98).

Common animal models used inperiodontal wound-healing research

A variety of different animal models, such as rats, dogsand nonhuman primates, have been used in peri-

odontal-regenerative studies (96, 128). The rat peri-odontal model has been frequently employed forbone-regeneration studies (49, 54, 57, 67). It is valuableas a screening model for assessment of regenerativemolecules because of cost-effectiveness, ease of han-dling, etc. However, the typical defect size is relativelysmall, making visualization challenging and requiringthe use of surgical microscopes for defect creation(129). Large animal models, such as the canine or thenonhuman primate, are a logical next step. The caninewound-healing kinetics and tooth anatomy havemany similarities to the human situation (143, 144).Nonhuman primates are highly desirable for evaluat-ing the safety and efficacy of new molecules becausetheir anatomic and biologic features are very similar tothose of humans (68, 128). However, their high costand handling difficulties prevent them from being

Table 2. Advantages and disadvantages of large-animal defect models

Defect type Animal model Advantages Disadvantages

Furcation/infrabonyperiodontal defect

Dog and monkey Surgical acute/chronic� short time to be created – less

expensive� standardized morphological

characteristics� does not regenerate spontaneously

(chronic)� Class II–III furcation� bilateral similar defect� horizontal defect allows an

estimation of the origin of the newlyformed tissue

� solid database describing healing (dog)� minimal palatal recession (monkey)

Surgical acute� does not reproduce inflammatory/

infective conditions� spontaneous partial regeneration

(monkey)Surgical chronic� soft tissues compromised� variable amount of connective

tissue regeneration

Ligature induced� microbiological features similar

to those of humans� morphological features similar

to those of humans� does not regenerate spontaneously� can make similar lesions as

control in contralateral defects

Ligature induced� nonpredictable disease

development� nonstandardized defect

morphology (dog)� requires time to be created

and is expensive

Alveolar socket Dog and monkey Easy and fast to performReproduces well the eventsoccurring in bone healing

Rapid bone repair compared withthat for human (dog)

Infrabony peri-implantdefect

Dog and monkey Surgically created� short time needed to generate

the defect� standard morphology-dimension

Ligature induced� morphological and microbiological

similarities with humans

Surgically created� spontaneous regeneration

Ligature induced� spontaneous partial regeneration� long time required to generate

the defect

Supra-alveolar peri-implantdefect

Dog Limited spontaneous regenerationReproducibly created

Requires space-providing devices

Reproduced from Pellegrini et al. (96), with permission.

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utilized more frequently. The preferred animal modelshould be selected according to study requirements,such as periodontal, oral implant, sinus floor augmen-tation and alveolar ridge reconstruction.

A study should not be proposed if a clear end-pointgoal is not established that will eventually have animpact on human health or disease. Animal-basedresearch has led to significant improvements in thequality of life for many patients (13, 95, 140). How-ever, these advances must result from the humaneuse and care of animals employed in such research.The field of periodontal regenerative medicine hasprogressed to provide better reporting and of designanimal studies with the aim of truly advancing thefield. The National Centre for the Replacement,Refinement and Reduction of Animals in Research, aUK Government-sponsored organization, has docu-mented overall marginal quality standards withrespect to the documentation, design and reportingof animal studies in research (65). These findingshave led to the development of guidelines for the

reporting of pre-clinical studies to improve their qual-ity and, overall, to reduce the use of animals inresearch. The ARRIVE (Animals in Research: Report-ing In Vivo Experiments) guidelines have been devel-oped using the CONSORT (Consolidated Standards ofReporting Trials) statement as their foundation (66).These guidelines have now become a requirement forthe publication of pre-clinical research in many sci-entific journals and support animal ethical bodies toimprove the overall quality of pre-clinical research.Furthermore, organizations, such as the EuropeanFederation of Periodontology, have addressed thisissue in several recent publications on periodontaland peri-implant wound healing (16, 122, 126, 139).

Future applications of periodontalwound-healing models

The use of pre-clinical animal models, typically undergood laboratory practice settings, is an importantcomponent in the development of new biomaterials

Fig. 2. The translational continuum from laboratory research discovery, through pre-clinical animal model testing andeventual acceptance to clinical practice. Reproduced from Quintessence Publishing, with permission [from Lin et al. (74),with permission].

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for clinical trials (118). In vivo models are, in general,superior to in vitro investigations in helping to under-stand the complex molecular, cellular and tissue reac-tions that occur in response to delivered regenerativemolecules. Despite the limitations of pre-clinicalmodels for human disease, in vitro investigations forthe simulation of human disease continue to remaininappropriate before testing in humans. Theincreased growth of development of biologics anddevices for application in periodontal-regenerativemedicine requires a thorough examination of whenand how the appropriate end points can be evaluatedbefore entry into human clinical trial testing. Withcontinued innovations in more noninvasive ex vivotechnologies, the needs for pre-clinical testing willonly decrease.

However, optimized animal models are still neces-sary for evaluating wound-healing promoters beforehuman testing. Limitations include obvious differencesin the host response and disease development, andanatomic differences among animals and humans.Models that can better implement risk factors of dis-ease development known to affect periodontal woundhealing, such as smoking, diabetes and microbialinfection, may aid in the continued refinement of pre-clinical animal models before embarking upon expen-sive human trials. As the field of personalizedmedicine continues to evolve, host susceptibility andidentification of those people who respond best towound-healing modifiers may provide more translat-able outcomes to individual patients. The ultimateresult of enhanced pre-clinical testing will greatlyadvance patient care for the public and aid in develop-ing a better understanding of wound-repair mecha-nisms.

What is the current ‘state of the art’in periodontal wound and bonehealing?

The first reviews in this volume of Periodontology2000 address the place for the various aforemen-tioned models employed to evaluate novel treatmentapproaches for periodontal, bone and peri-implantwound healing and their potential role in informingthe implementation of clinical therapy in humans(41, 60, 124, 132).

Pre-clinical studies are usually performed to eval-uate proof-of-principle concepts, safety and possi-ble unwanted effects of candidate biologic agentsor bone biomaterials, before proceeding into clini-cal testing (45, 86, 96). Selection of the appropriate

animal species and wound-healing model is largelydependent upon the research question or the dis-ease model. For example, models involving smallanimals have been developed largely to screenbone biomaterials for their potential to enhancebone formation. As no single model can completelymimic the anatomic, physiologic, biomechanic andfunctional environment of the human mouth andjaws, the results obtained in small animals need tobe validated in larger animals (e.g. dogs, mini-pigsor monkeys) whose dento–alveolar anatomy resem-bles more closely that of humans.

Following application of the guided tissue regener-ation principle for the treatment of periodontaldefects, the same principle was used in a number ofpre-clinical and clinical studies for the regenerationof bone defects or for the augmentation of deficientsites. For these techniques, the term ‘guided boneregeneration’ was adopted, pointing to the biologicrationale based on the mechanical exclusion ofneighboring soft tissues surrounding the bonedefects, in order to allow only osteogenic cells derivedfrom the margins of the bony defect to repopulate thewound area. This biologic principle, also based uponMelcher’s theories, was first tested in smaller animalmodels, such as rats and rabbits, and was confirmedlater in larger animals (e.g. dogs and monkeys) (4, 37–40, 70).

An important issue when testing the guided boneregeneration principle is the use of the so-called ‘criti-cal size defect’, indicating bone defects that healspontaneously with fibrous connective tissue and notby osseous fill. Critical-size defect models involvehealing of orthotopic bone sites, such as the mandibleor the calvarium, and thus allow the evaluation ofbone grafts and other bone-regenerative modalitiesin a standardized and discriminative manner to pro-vide ‘proof-of-principle’ (14, 38, 58, 70, 117, 118, 119,137). As it has been repeatedly demonstrated thatguided bone regeneration can successfully regeneratecritical size defects, nowadays the use of such experi-mental models for evaluating the effect of differenttreatment modalities for bone regeneration is recom-mended, as discussed by Donos et al. (41) in this vol-ume of Periodontology 2000.

The guided bone regeneration principle has beensuccessfully applied in the treatment of patientsexhibiting different types of bone defects, leading to asubstantial increase in the number of cases of dentalimplant therapy, including cases that were previouslynot feasible because of a lack of bone at the recipientsite (23–26, 51, 92). Taken together, these animalexperiments serve as a beautiful example of the

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importance of pre-clinical models (Phase 1 studies) intranslational regenerative dental research.

The successful integration of guided bone regen-eration principles into clinical practice over the lasttwo decades has led to a substantial increase inthe number of dental implants being placed andassociated bone-augmentation procedures. This hascoincided with an ageing population whose expec-tations of tooth replacement with fixed prostheseshave also increased (81, 131). However, there hasbeen a coincident increase in the proportion of thepopulation with age-related chronic diseases andconditions, such as diabetes and osteoporosis (50,69, 104). Their impact upon bone-regeneration pro-cedures related to implant rehabilitation is largelyunexplored and consequently there is a pressingneed to evaluate different bone-regenerative tech-niques in pre-clinical models, to help optimize thetreatment of patients affected by such systemicconditions. Donos et al. (41) discuss these issues intheir review. Interestingly, recent data from preclin-ical models indicate that, in osteoporotic animals, ahydrophilic barrier/implant surface may counteractthe negative effect of osteoporotic-like conditions,whilst the use of guided bone regeneration appearsto create an environment that facilitates bone for-mation in experimentally induced diabetes andosteoporosis (73, 79). Despite the fact that, at pres-ent, it is difficult to draw any conclusions regardingthe extent to which systemic conditions may affectbone metabolic status, vascularization and healingcapacity, the use of various pre-clinical models simu-lating systemic health and disease are of utmost clin-ical relevance for developing new concepts thatenable de novo bone formation via guided boneregeneration in medically compromised patients.

The extraction socket as a wound-healingmodel

Tooth extraction is one of the most common surgicalprocedures performed on humans, yet reviewing dec-ades of published literature it is remarkable to notethat, until recently, very little was known about thebiologic processes that take place during socket heal-ing. Studying the simplest of wound-healing models(i.e. the extraction socket) has provided profoundinsights into the temporal sequence of healing eventsthat follow tooth extraction (6, 10, 11, 29, 74).

Studies by Araujo et al. (8) have demonstrated thatthe overall wound-healing events described histologi-cally in dogs are remarkably similar to those inhumans, despite the fact that bone modeling and

remodeling during socket healing are three- to fivetimes quicker in dogs. Such findings subsequently ledto the development of clinical protocols associatedwith tooth extraction. For example, it is currently rec-ommended that tooth extraction is planned and per-formed with attention paid to the subsequentanticipated changes in ridge width and height follow-ing this intervention. As a result, there is frequentlyan indication to consider adjunctive therapies inorder to compensate for such changes, particularly incases in whom further treatment, such as dentalimplant placement, is planned (9, 75).

Pre-clinical and human models to studyosseointegration and management ofperi-implant infections

The use of pre-clinical (animal) and human modelshas been shown to provide valuable informationregarding our understanding of the healing of hardand soft tissue integration around titanium dentalimplants (1–3, 18, 42, 71, 85). Such healing resultsfrom a complex cascade of biologic events that areinitiated by the surgical intervention. The temporalsequence of healing events that culminate in osseoin-tegration has been elucidated in animals andhumans. Implant placement into alveolar boneinduces a cascade of healing steps, commencing withclot formation and continuing with bone maturationin direct contact with the implant surface. Salvi et al.(113) demonstrate that osseointegration is associatedwith a decrease in inflammation and an increase inosteogenesis-, angiogenesis- and neurogenesis-related gene expression during the early stages ofwound healing. The attachment and maturation ofthe soft-tissue complex to implants (i.e. epitheliumand connective tissue) is established 6–8 weeks fol-lowing surgery (126). Moreover, the use of pre-clinicaldata has provided important insights into the etiol-ogy, pathogenesis and therapeutic approaches toperi-implant diseases because established lesions inanimals have shown many features in common withthose analyzed in human biopsy material (5, 27, 28,121–123).

The review of Romanos (108) provides some evi-dence that immediate loading of augmented ridgesmay improve bone stability following implant place-ment, and Schwarz et al. (124) discuss the utility ofanimal models in developing therapeutic approachesto implant failure (97, 109–111, 124). Renvert & Polyz-ois (103) discuss the limitations of existingapproaches to managing peri-implant mucositis andperi-implantitis in humans and conclude that the

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most important factor in managing implant failure ispatient self-performed oral hygiene.

What can in vitro models offer for abetter understanding of periodontalwound and bone healing?

In order to improve and further develop treatmentstrategies aiming to regenerate soft and hard tissuesat natural teeth and dental implants, it is importantto acquire a thorough understanding of the charac-teristics and roles of different wound-healing compo-nents (such as cells, matrices, molecules and genes)involved in the complex processes of wound healingand repair, as well as the mechanisms controllingtheir function. As these processes are complex, it isextremely difficult to understand and define the roleof each component in determining the final outcome.Furthermore, in animal models that aim to simulate acertain clinical situation, the influence of individualcomponents in the wound-healing cascade is difficultto assess. Such models are also expensive andfrequently difficult to work with. Accordingly, in orderto understand a certain mechanism, or before testinga specific biologic principle or biomaterial in animals,it makes sense to adopt a stratified approach byanswering single, well-defined questions using specif-ically designed laboratory models.

A plethora of in vitro models have been describedand developed to mimic the wound-healing events ofsoft tissues (e.g. oral epithelia or oral fibroblasts) or tomeasure proliferation, attachment and migration,gene expression and differentiation into a minerali-zing phenotype, as well as biomineralization ofperiodontal ligament fibroblasts or progenitors, osteo-blasts or osteoprogenitors and cementoblasts. Otherin vitro studies have utilized various gene-transfermodels, models designed to evaluate the effect ofimplant surface modifications on cellular behavior ormodels that address biocompatibility and degradationof different types of resorbable membranes (34, 142).

Despite the fact that, in general, such laboratory-based models are less expensive and easier to per-form compared with animal experiments, they differmarkedly from the in vivo situation because they typ-ically employ single cell types without the surround-ing matrix or other relevant cell types, and withoutthe influence of external factors. Therefore, resultsobtained using such models often provide criticalmechanistic and biologic plausibility insights. Thesestudies lay the foundation for potential clinical appli-cation in more applicable pre-clinical and clinicalstudies on the disease and injury processes.

Improved clinical outcomes through animproved understanding of biology

There is little doubt that research conducted overthe last 25 years into the biophysiology of periodon-tal and tooth-socket healing has informed the devel-opment of novel approaches to restoring lostperiodontal tissues and bone (8, 44, 62, 125). In thelaser field, comprehensively reviewed by Aoki et al.(7), a greater understanding of the role of certainwavelengths in removing soft and hard deposits, kill-ing bacteria and improving wound healing, has ledto the application of lasers and of laser-based photo-dynamic therapy in clinical periodontal care. Somepositive effects, such as the reduction of inflamma-tion (i.e. less bleeding on probing), following theadditional application of photodynamic therapy,have been demonstrated, in randomized controlledclinical studies, to be statistically significant and clini-cally relevant, and consequently such approaches arefrequently used in the maintenance phase (15, 35,116, 136). By contrast, it is clear that laser therapyhas thus far failed to demonstrate improved peri-odontal outcomes over and above those achieved bytraditional instrumentation (120). It is also importantto report such negative findings because they savethe effort and costs involved in repeating clinical tri-als of the same, and open up opportunities toexplore the utility of different wavelengths and pro-tocols.

Other examples of tissue-regeneration techniquesthat have been developed based upon learnings frombiologic research and have improved clinical out-comes, include intrabony defects, furcation lesions[see reviews by Cortellini & Tonetti (36), and Sanzet al. (114)] and recession defects [see Zucchelli &Mounssif (145)]. The novel concepts reported in thesereviews have evolved following in vitro and animalstudies, which have informed a better understandingof periodontal and bone wound healing and of therole and use of various biomaterials on periodontalligament cells, gingival fibroblasts and bone cells.These techniques combine the use of surgical flapsand suturing techniques [see Burkhardt & Lang (22)]designed to improve blood clot protection and toenhance wound stability, and the use of biologicallypotent biomaterials.

Stem cell research holds great promise for the futureof periodontal regeneration, but multiple challengesremain, including the development of appropriatematrices to deliver cell therapies, appropriate growthfactor combinations that facilitate physiological heal-ing processes in what is ultimately a potentially ‘infec-

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tive’ healing environment. The latter is a challengethat is frequently overlooked or underestimated, butinvolves stimulating the body’s physiological wound-repair processes within a complex milieu of microbialand host signaling between innate and acquiredimmune systems and a colonizing microbiome. Simi-lar challenges face bone-regenerative proceduresaround failing implants, which lack a periodontal liga-ment (and thus a vital source of undifferentiated stemcells) and also differ in terms of the connective tissuefiber anatomy within the investing soft-tissue cuff.Here, emerging evidence indicates that early out-comes of bone regeneration are more predictablewhen the implant fixture is recovered by soft tissues tofacilitate healing in a ‘closed system’, in which micro-bial colonization is not a complication. Nevertheless,longer-term studies are needed in this emerging fieldin order to achieve a better understanding of the biol-ogy of healing around implants that have sufferedperi-implantitis and also to understand the nature ofthe primary process of peri-implantitis at the tissuelevel. It appears that we are now at the end of thebeginning of research into periodontal and boneregeneration, and understanding the pathobiology ofperiodontitis and peri-implantitis is fundamental toenable the design of regenerative strategies and tech-nologies, rather than persisting with the genericapproaches of today.

Acknowledgments

The collaborations from Qiming Jin, Jim Sugai, HectorRios, Reinhard Gruber, Yang-jo Seol, Gaia Pellegrini,and Darnell Kaigler are greatly appreciated. Dr Gian-nobile’s work was supported, in part, by NIH/NIDCRDE 13397.

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