Periodontology 2000, Val. 19, 1999, 164-1 72 Printed in Denmark .

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C o p y r i g h t 0 Munksgaard 1999

PERIODONTOLOGY 2000
ISSN 0906-6713

Infectious aspects of periodontal regeneration

J 0 R G E N SLOTS, ERIKA M I T H MACDONALD HESSAM S & NOWZARI The development of predictable procedures to facilitate the formation of new periodontal ligament, cementum and bone over previously diseased root surfaces remains an important goal in periodontal therapy. Periodontal regeneration has been attempted using extraoral or intraoral autogenous bone grafts, allografts, various alloplasts, growth factors, nonresorbable or resorbable barrier membranes, or their combinations (2). Regenerative approaches to treat periodontitis lesions offer exciting possibilities; however, they frequently fail to produce the desired clinical outcome due to infectious complications (27). Because regenerative periodontal devices are placed in a potentially highly infected environment, their successful application depends upon the prior removal or marked suppression of microbial pathogens at the treated site(s). Infection control may be particularly important with barrier membranes because they may induce a foreign body-like reaction with reduced host defenses ( 5 3 ) . This chapter reviews the microbiota that may interfere with periodontal regeneration. It also provides guidance on antimicrobial therapies that may reduce the risk of infectious failures with periodontal regeneration. Emphasis is placed on infections associated with expanded polytetrafluoroethylene barrier membranes because most of the available data are concerned with this type of regenerative device. rier membranes of different materials. Wang et al. (50) compared the in vitro attachment of 15 oral microorganisms to expanded polytetrafluoroethylene, polyglactin 910 and collagen barrier membranes. Streptococcus mutans revealed the strongest attachment in all experiments except for Porphyromonas gingivalis, which exhibited higher cell counts on collagen membranes. S. mutans attached significantly more to the expanded polytetrafluoroethylene and the collagen barrier membranes than to the polyglactin 9 10 membrane. Actinobacillus actinomycetemcomitans, Actinomyces viscosus, Fusobacterium nucleatum and Selenomonas sputigena showed similar attachment to the 3 barrier membranes studied. S. sputigena revealed the lowest attachment potential of the test bacteria. Ricci et al. (34) demonstrated varying in vitro affinity for J? gingivalis to attach to 6 types of bioabsorbable and nonbioabsorbable barrier membranes. A polyglactin and two collagen barrier membranes demonstrated high I? gingivalis counts. Polylactic acid and synthetic glycolide and lactide copolymer barrier membranes showed lowest J? gingivalis counts. Expanded polytetrafluoroethylene barrier membranes revealed I! gingivalis cells mainly in the fibrillar areas of both the internal and external sides of the membrane. Ricci el al. (34) also showed the in vitro ability of I? gingivalis to penetrate barrier membranes from one side to the other. Simion et al. (40) reported that an expanded polytetrafluoroethylene barrier membrane had to be exposed for at least three weeks for complete bacterial penetration. Using scanning electron microscopy, Selvig et al. (38) identified cocci, filaments and short curved rods in the outer and inner surfaces of retrieved barrier membranes. The patients had been prescribed tetracycline for 2 weeks postsurgically and chlorhexidine rinses during the period of membrane insertion. Selvig et al. (39) showed an inverse relationship between the extent of barrier membrane microbial contamination and gain in clinical attachment level.

Microbiota of failing periodontal regeneration

Table 1 presents studies that have examined the effect of microbial biofilms or specific bacteria on regenerative periodontal therapy. Oral bacteria have a propensity to colonize barrier membranes (26, 38). Moreover, in vitro data suggest that bacterial species vary in ability to attach to bar-

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Grevstad & Leknes (14) detected gram-negative rods and spirochetes in the occlusive and apical portions of expanded polytetrafluoroethylene barrier membranes despite systemic penicilllin therapy and chlorhexidine rinses. The authors attributed the microbial growth on barrier membranes to ineffective antimicrobial therapy. Chen et al. (4) compared the rate at which 11 common oral bacterial species colonized expanded polytetrafluoroethylene, collagen and polylactic acid barrier membranes in vivo. Total bacterial attachment increased over the 2-week study period, but the 3 barrier types did not differ in numbers or species of colonizing bacteria. Tempro & Nalbandian (45) recovered predominantly Streptococcus and Actinomyces species and occasionally Haemophilus and Capnocytophaga species from the microstructure and the occlusive portions of expanded polytetrafluoroethylene barrier membrane. They proposed that barrier membrane infections may in part be responsible for the varying clinical outcome with guided tissue regeneration. De Sanctis et al. (8, 9) evaluated bacterial colonization of expanded polytetrafluoroethylene and polyglicolactic barrier membranes at 4-6 weeks after membrane insertion. In scanning electron microscopy, 57% of exposed and only 17% of nonexposed barrier membranes revealed bacteria in the mid-part of the tooth-facing surface of the barrier membrane (8). De Santis et al. (8) associated bacterial colonization in the mid-part of the inner surface of the barrier membrane with an almost 50% reduction in potential gain of clinical attachment. Nowzari & Slots (25) demonstrated an inverse relationship between microbial counts on expanded polytetrafluoroethylene barrier membranes and gain of probing attachment in treatment of furcation and two- to three-wall intraosseous periodontal lesions. Eighty percent of teeth having membranes with less than lo8 total viable counts gained 3 mm or more in probing attachment, whereas teeth with membranes harboring more than lo8 total viable counts either lost attachment (50%)or showed increases in attachment of only 1 or 2 mm. Mombelli et al. (21) recovered specific bacterial species from barrier membranes retrieved from periodontal sites showing 1-5 mm gain in clinical attachment. The patients performed strict plaque control and rinsed with chlorhexidine but received no systemic or topical antibiotic therapy. Gramnegative anaerobic rods made up 31% of total membrane isolates at 6 weeks postsurgery. One site yielded high proportions of I? gingivalis, 6 sites dem-

onstrated Prevotella intermedia and 6 sites showed Prevotella melaninogenica. Fusobacterium and Capnocytophaga species were also frequent membrane isolates. No analysis was performed to determine the possible role of specific microorganisms on clinical outcome. Specific bacteria seem to assume particular importance in failing periodontal regeneration. Nowzari & Slots (25) detected I? gingivalis in 3 periodontal sites that experienced a net loss of clinical attachment after membrane removal. A. actinomycetemcomitans was recovered from a periodontal site that gained as little as 1 mm of probing attachment. Campylobacter rectus comprised 14%of the membrane microbiota at a tooth that demonstrated 1 mm gain of clinical attachment. Total microbial counts and percentage of Peptostreptococcus micros, Capnocytophaga species and motile rods on barrier membranes seemed also negatively to affect periodontal regeneration. Nowzari et al. (28) found most gain in clinical attachment in sites that showed no periodontal pathogens on the tooth-facing surface of the barrier membrane. Sites exhibiting little or no gain in clinical attachment demonstrated several species of periodontal pathogens on both sides of the barrier membrane. Machtei et al. (17) implicated A. actinomycetemcomitans in failing regenerative periodontal therapy. Demolon et al. (10) found an association between I? gingivalis and Bacteroides forsythus on polytetrafluoroethylene barrier membranes and clinical signs of inflammation. Microbial colonization of barrier membranes may already start at the time of membrane insertion (29). Also, periodontal pathogens that contaminate a barrier membrane during insertion may persist on the membrane for a prolonged period (29). Pathogen colonization of barrier membranes during the initial intraoral manipulation has been associated with reduced gain of clinical attachment (29). Bacteria colonizing barrier membranes during the time of insertion probably originate from saliva or, in case of insufficient debridement, from the membrane-treated lesion itself. Patients having high subgingival counts of periodontal pathogens in periodontitis lesions other than the site receiving regenerative therapy exhibit an increased risk for barrier membrane colonization by motile rods, I? gingivalis, I? intermedia, I? micros, Propionibacterium species and spirochetes (29). Most likely, the membrane infecting pathogens are transferred via saliva from infected periodontal lesions or tongue dorsum to the regenerative-treated periodontal site(s). Herpesviruses have recently been implicated in

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Table 1. Relationship between barrier membrane biofilm and failing periodontal regeneration
-____

-___

Reference Noppe, Wachtel, Bernimoulin, Ebert-Kayser (23)

Type of membrane~Type of periodontal defect Plaque analysis- Microbiological findings and clinical outcome -~ ~40 intraosseous Retrieved Expanded Histologically, all exposed parts of the barrier membrane were polytetrafluoroethylene contaminated by microorganisms, which were found bedefects membranes tween neutrophds, degenerated collagen fibrils and necrotic for light and cell components. Clinically, most cases showed membrane extransmission posure with gingival recession and pocket formation between electronic membrane and gingiva. microscopy
_ _
-

Selvig, Nilveus, Fitzmorris, Kersten, Khorsandi (38) Expanded polytetrafluoroethylene scanning electron microscopy Expanded polytetrafluoroethylene Class I1 furcation defects Anaerobic culture

-___ 14 intraosseous and Retrieved 6 furcation defects membranes for

Bacterial colonies extended into the midpart of the membrane. Fibroblast-like cells, blood vessels and fibrous structures were also found. The occlusive portion of the membrane had a sparseness of adherent tissue elements. No correlation was made between microbiological and clinical findings. Microbiological data underscored the significance of surgical instrumentation in inter-radicular treated sites and confirmed the need to maintain healing sites free of periodontal pathogens.

Valletta, Sbordone, Rarnaglia, Ciaglia, Spagnuolo, Lenci (48)

Sbordone, Ciaglia, Spagunuolo, Lenci, Barone, Ramaglia (37)

1 nonabsorbable and 1 absorbable 2 periodontal defects
__ 12 intraosseous defects
~

Not known

Selvig, Kersten, Chamberlain, Wikesjo (39)

Expanded polytetrafluoroethylene

No clinical difference between type of membranes employed. The authors suggested that healing sites should be free of periodontal pathogens. Retrieved Comparisons between ultrastructural findings and clinical obmembranes servations revealed that the extent of bacterial colonization for scanning of the membrane correlated inversely with clinical attachment electron microscopy gain. Extent of oral exposure may be an indicator of long-term success or failure of guided tissue regeneration.
30 retrieved membranes by scanning electron microscopy
4 partially exposed

Guillemin, Mellonig, Brunsvold (15)

__
~.

Expanded polytetrafluoroethylene

30 intraosseous defects

No clear pattern of microbial colonization or cell adherences in either side of the membrane. The presence of plaque on the membrane did not hamper clinical healing during the first 4-6 weeks.
3 structurally different groups of bacterial aggregations were observed 1) gram-positive cocci and rods in the external part of the open microstructure, 2) cocci, rods and filamentous microorganisms embedded in fibrin-med spaces, and 3) gram-positive cocci and rods, gram-negative microorganisms and spirochetes in the occlusive part of the membrane. No correlation was made with clinical outcome.

Grevstad, Leknes (14)

Expanded polytetrafluoroethylene

8 intraosseous periodontal defects

membranes by electronic microscopy

Tempro, Nalbandian (45)

Expanded polytetrafluoroethylene

6 periodontal intraosseousdefects and 2 furcation defects

6 retrieved membranes for transmission electron microscopy and anaerobic culture

Demolon, Persson, Moncla, Johnson, Ammons (10)

Expanded polytetrafluoroethylene

19 class I1 furcation defects

Scanning electron microscopy

Transmission electron microscopy showed numerous bacteria including cocci, rods and filaments. Anaerobic culture yielded Streptococcusand Actinomyces species, and gram-negative facultative rods, mainly Huernophilus species. It was assumed that bacterial colonization of membrane affects periodontal connective tissue regeneration. __ Augmentin" group showed less barrier-membrane exposure than controls. B. jorsythus and E! gingivulis were associated with gingival inflammation during initial healing. Augmentin@ seemed to have little effect on barrier-membrane microbiota.

Table 1. Continued
Expanded polytetrafluoroethylene 11 2- to 3-wall and furcation defects Selective and non-selective anaerobic culture and DNA probe

Nowzari, Slots (25)

Novaes, Gutierrez, Francischetto, Cellulose Novaes (24)

10 class I1 furcation defects

ari, Matian, Slots (28) Expanded polytetrafluoroethylene 10 angular bony defects

10 angular bony defects

18 two- to three-wall intraosseous defects

De Sanctis, Zuccheli, Clauser (9) Expanded polytetrafluoroethylene Zuccheli, Clauser (8) Polyglycolactic acid

Machtei, Grossi, Dunford, defects 10 mandibular furcation defects

Expanded

56 Class I1 furcation

Nowzari, Smith MacDonald, London, Flynn, Morrison, Slots (29)

2- to 3-wall defects

Expanded polytetrafluoroethylene

42 mandibular posterior

onald, Nowzari, Flynn, Morrison,

20 2- to 3-watl defects Polylactic acid and expanded polytetrafluoroethylene

Inverse relationship between microbial counts and gain of probing attachment. 80% of teeth having membranes with less than lo8 total viable counts gained 3 mm or more in attachment; teeth with membranes having more than lo8 bacteria lost or had small probing attachment increases of 1-2 mm. Scanning electron Scanning electron microscopy and DNA probe analyses did microscopy and not reveal periodontal pathogens at the time of membrane DNA probe exposure. No clinical results were reported. Selective and The Augmentine group had an increase in mean probing non-selective attachment of 36.5% of potential gain as compared with anaerobic e 22.4% for control group. The Augmentin@ group showed 9fold fewer organisms than controls. and DNA Bacterial colonization in the mid-part of barrier membrane Scanning electron reduced potential probing attachment by 50%. microscouv Gain of probing attachment was greater for non-exposed than Scanning electron for exposed membranes (4.220.5 mm vs 3.3k0.6 mm respecmicroscopy tively). Scanning electron microscopy showed that 41% of exDosed membranes had bacterial colonization. Sites colonized with l? intermedia exhibited less favorable Anaerobic culture clinical results compared with sites free of the organism. Fusobacterium, I! intermedia and Actinomyces odonsolyticus Anaerobic culture were most common membrane isolates. Sites treated with a o be positive for 15 target microbamer membrane ten organisms more often sites treated without a membrane. Selective and Patients treated with hll-mouth osseous surgery revealed non-selective lower levels of periodontal pathogens on barrier membranes anaerobic culture and greater gain in clinical attachment than controls (3.4 us 1.4 mm respectively). and DNA probe Selective and nonNo statistically significant difference was found between the selective anaerobic two barrier membranes in microbiological and clinical findculture, DNA probe, ings. For every 10%pathogen level at barrier-treated sites, nested polymerase 0.43k0.23 mm less gain in clinical attachment level was obchain reaction for served. Virally positive barrier-treated sites exhibited 2.520.9 herpesvirus detection mm less gain in clinical attachment than virally negative barrier-treated sites.

Slots et al.

failing periodontal regeneration. Smith MacDonald et al. (44) studied 4 membrane-treated periodontal sites having human cytomegalovirus or Epstein-Barr type 1 virus. The 4 virally infected sites revealed an average gain in clinical attachment of 2.3 mm compared with 16 virally negative sites that showed a mean clinical attachment gain of 5.0 mm (P=0.004). This observation may be important because the two herpesviruses have been associated with adult periodontitis (5, 31) and with acute necrotizing ulcerative gingivitis in Nigerian children (6) and they possess an array of virulence factors pertinent for destructive periodontal disease (5). It is also of interest that cytomegalovirus is the most common viral infectious complication in patients receiving organ and bone marrow transplants (35). Cytomegalovirus can infect and alter the function of fibroblasts (201, thereby potentially impairing regeneration of the periodontal ligament. More research is warranted on the possible relationship between periodontal herpesvirus infections and suboptimal healing after regenerative periodontal procedures. Thus, it seems that several oral microbial species can colonize barrier membranes, that microbial contamination of barrier membranes can significantly hamper periodontal tissue regeneration, and that certain microbial species and maybe some herpesviruses are particularly detrimental to periodontal regeneration.

Anti -infective therapy in periodontal regeneration

To prevent infectious failures with regenerative periodontal therapy, efforts should be directed towards the elimination or marked suppression of pathogens in the treated site as well as in the entire mouth prior to regenerative surgery and throughout the healing phase. The dental professional possesses a variety of means to prevent or control barrier membrane-associated infections. Anti-infective therapy in periodontal regeneration includes mechanical debridement with or without concomitant periodontal surgery, local or systemic delivery of antimicrobial agents, possible application of slow-release antimicrobial agents to the barrier membrane material and proper oral hygiene measures by the patient. Diligent mechanical instrumentation (that is, sealing and root planing) to remove dental plaque, calculus, altered cementum and cytotoxic microbial products on the root surface is a prerequisite for op-

timal periodontal healing. Patients may also benefit from treatment of periodontal lesions other than those designated for regenerative therapy before performing the regenerative procedures. Nowzari et al. (29) showed that patients who received pocket-reduction therapy of existing periodontal lesions prior to barrier membrane placement revealed the lowest levels of periodontal pathogens in the membrane and exhibited most clinical attachment gain. A nonsurgical approach to periodontal therapy may also be successful in reducing the oral load of periodontal pathogens, provided appropriate considerations are given to the specific pathogenic microbiota of the patient. As discussed by van Winkelhoff et al. (49), systemic antimicrobial therapy may be necessary to suppress A. actinomycetemcomitans and other periodontal pathogens in the human oral cavity. Systemic antimicrobial treatment allows antibiotics to reach pathogens residing in the depth of periodontal pockets and on the tongue and buccal mucosa. Systemic administration of amoxicillin-clavulanic acid (Augmentin9 (28) and ornidazole (22) have demonstrated clinical benefits in regenerative periodontal therapy. However, since an antibiotic agent may be active against one and demonstrate resistance against another periodontal pathogen and since the periodontal microbiota of individual patients often shows several pathogenic species simultaneously, it is often advantageous to prescribe a combination therapy of two antibiotics. Metronidazole and ciprofloxacin exhibit synergy and constitute a valuable drug combination for periodontitis patients with anaerobic pathogens together with superinfecting enteric bacteria (32, 42). The metronidazole-ciprofloxacin drug combination is also effective against A. actinomycetemcomitans and several other periodontal pathogens (42). Resistant organisms are mainly streptococci (421, which exert antagonistic activity against several periodontal pathogens (42, 49). Optimally, systemic antibiotics in periodontics should be prescribed on the basis of an appropriate microbiological analysis (41, 46). If mere clinical judgment forms the basis for the selection of antibiotics, the clinician may not choose the optimal drug regimens and may not obtain the desired clinical results (43). Nowzari et al. (29) recommended washing extraoral skin areas with a iodophor before regenerative periodontal surgery to prevent membrane colonization by dermal pathogens during membrane insertion. Sutures used in regenerative procedures are particularly prone to contacting the lips and the face and thereby adsorbing dermal microorganisms.

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Infectious aspects of periodontal regeneration

Sander et al. (36) evaluated the effect of local application of 1 g of a 25% metronidazole gel on periodontal healing after guided tissue regeneration. At 6 months postsurgery, significantly more gain of clinical attachment occurred in the metronidazoletreated periodontal sites than in sites treated with barrier membrane alone (36).At 1 week postsurgery, metronidazole-treated sites showed significantly lower levels of total viable bacteria and lower proportions of black-pigmented anaerobic rods than control sites (12). However, no microbiological difference between test and control sites was observed at later observation times (12), suggesting that the microbiological benefit of the metronidazole gel was confined to the initial regeneration phase. Local pocket delivery of chemotherapeutic agents other than metronidazole may also help reduce pathogens in barrier membrane-treated sites (33). Antimicrobial coating of barrier membranes constitutes a novel approach to controlling membraneassociated infections. Harvey et al. (16) have described the potential of antibiotic bonding to polytetrafluoroethylene by means of the cationic surfactant, tridodecylmethylammonium chloride (Polysciences, Inc., Warrington, PA, USA). Tridodecylmethylammonium chloride has been safely used for more than a decade in vascular shunt (3) and urinary tract catheter (11) surgeries. Our laboratory has studied the clinical benefit of applying tetracyclineHC1 to the surface of expanded polytetrafluoroethylene membranes (30).Barrier membranes were submerged for 1 minute in a 5% solution in absolute alcohol of tridodecylmethylammonium chloride. After drying at room temperature, the specimens were immersed for 1 minute into a freshly prepared, basic 3% tetracycline-HC1solution (one 250 mg capsule dissolved in 7.5 ml distilled water; pH adjusted from 1.7 to 9.5 using sodium hydroxide) and then dried at room temperature. In vitro studies showed that, despite exhaustive washing, the tetracyclinecoated barrier membranes released biologically active tetracycline for 3-6 weeks (Fig. 1). Tetracyclinecoated barrier membranes harbored significantly fewer cultivable pathogens for the first 2 weeks after membrane insertion than non-coated membranes (30). Also, after 6 weeks, tetracycline-coated membranes were associated with more gain in clinical attachment than non-coated membranes (30).Most dental offices can readily perform the tetracycline coating described above. Undoubtedly, additional methods for slow release of antibiotics or other biologically active agents from regenerative devices will be developed in the future.

Fig. 1. Tetracycline coating of a section of polytetrafluoroethylenebarrier membrane by means of tridodecylmethylammonium chloride. After 21 days of washing in double distilled water with frequent water changes, the membrane still released active tetracycline, as evidenced by the zone of inhibition of Escherichia coli growth around the membrane.

The optimal supportive periodontal treatment after regenerative periodontal therapy has not been firmly delineated, and treatment may have to be modified to fit specific individual needs. However, most anti-infective principles of conventional periodontal maintenance therapy are applicable to regenerative procedures (52). Proper oral hygiene is important to decrease plaque levels for the benefit of the tissue healing. Studies have shown regenerative periodontal procedures to be more successful in patients having little or no dental plaque than in patients with high plaque levels (47).Also, patients with good oral hygiene can preserve the clinical attachment gain after regenerative periodontal therapy for many years (7, 13, 18, 19, 51). Commonly, patients are instructed in twice-daily use of toothbrushing and dental flossing. Chlorhexidine rinses seem to offer therapeutic benefits during healing (211, even though in uitro experiments have revealed an ability of chlorhexidine to damage fibroblasts (1). Some clinicians advocate supportive periodontal treatment once a week for the first 6 weeks of barrier membrane treatment and then once a month for the first 6 months after regenerative therapy (29). In barrier membrane-treated sites, mechanical plaque removal should be performed in a gentle manner in order not to disturb tissue healing. Also, to avoid a foreign body-like reaction, pumice or other particular matter that may remain in regenerative sites during the healing phase should be used carefully and sparingly.

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Slots e t al.

Recommendations and concluding remarks

Regeneration of periodontal tissue is predicated upon controlling periodontal pathogens in the barrier membrane-treated site and probably also in other parts of the oral cavity prior to the membrane insertion and during initial healing. I! gingivalis, A. actinomycetemcomitans, B. forsythus, I? intermedia, l? micros and C. rectus have been associated with failing regenerative periodontal therapy. Herpesviruses may play a detrimental role in periodontal regeneration as well. Topical antiseptic and systemic antibiotic therapy might be employed to control pathogens in periodontal regeneration. A practical approach to antimicrobial therapy in regenerative periodontal therapy is outlined below: institution of effective oral hygiene measures; subgingival scaling and root planing of the entire dentition, possibly combined with periodontal surgery to gain access to affected root surfaces; surgical reduction of periodontal pockets (except for the site(s) of regenerative surgery) or appropriate nonsurgical therapy of periodontally affected teeth; possible utilization of a tetracycline-coated barrier membrane; thorough root planing of barrier membranetreated sites using ultrasonic, rotary or hand instrument; application of antimicrobial agent to diseased root surfaces (iodophor, metronidazole, tetracycline or citric acid, but probably not chlorhexidine due to its toxic effect on fibroblasts); and suturing of surgical flaps for complete coverage of the barrier membrane; possible systemic antibiotic therapy with an appropriate antimicrobial agent to eradicate or markedly suppress periodontal pathogens. Antibiotic therapy may begin 1-2 days prior to regenerative surgery and continue for at least 8 days for most antibiotics; 0.12% chlorhexidine rinse (15 ml, twice daily) during the first 2 weeks after regenerative surgery; the anti-inflammatory ibuprofen (400 mg, four times daily) may be prescribed for 3 days postsurgery; frequent professional plaque removal during the healing period. Supportive periodontal treatment may be scheduled once a week for 6 weeks and then once a month for 6 months. Subgingival scaling, probing and pumicing of regenerative sites should be minimized or avoided for the first 6

months not to disturb the maturing of newly formed connective tissue; and institution of an individually tailored maintenance care program. This chapter has focused on infectious aspects of membrane-assisted periodontal regeneration. However, the concept of infection control is applicable also to other types of regenerative periodontal therapy and to guided bone regeneration. It is important to realize that the formation of new connective periodontal attachment is contingent upon the elimination or marked reduction of pathogens at the treated periodontal site.

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