Loss of claudin-1 in lipopolysaccharide-treated periodontal epithelium.

BACKGROUND AND OBJECTIVE: The epithelial barrier is a critical component of innate immunity and provides protection against microbial invasion. Claudin-1, a tight junction protein, is known to contribute to the epithelial cell barrier. An experimentally induced rat periodontal disease model was used to study the effects of lipopolysaccharide (LPS) on the expression of tight junction-associated molecule genes in the junctional epithelium. MATERIAL AND METHODS: LPS was applied for 8 wk in the gingival sulcus, and junctional epithelium was collected by laser-capture microdissection and subjected to microarray analysis. RESULTS: Microarray analysis identified that expression of the claudin-1 gene was decreased in the epithelium by chronic LPS challenge. Immunohistochemical analysis confirmed the expression of claudin-1 protein in junctional epithelium and that 8 wk of chronic LPS topical application significantly reduced claudin-1 expression. The effect of LPS on claudin-1 protein expression was validated using a porcine junctional epithelial cell culture Transwell model. The epithelial barrier, as measured using transmembrane resistance, was significantly reduced after 3 wk of LPS challenge and this was associated with a decreased level of expression of claudin-1 protein. CONCLUSION: These results confirm that the initiation of experimental periodontal disease is associated with reduction in the expression of claudin-1 gene and protein. This decreased level of a critical tight junction protein may result in the disruption of barrier function and may play an important role in the initiation of periodontal disease.

Microtopographical regulation of adult bone marrow progenitor cells chondrogenic and osteogenic gene and protein expressions.

Microtopographic features affect diverse cell behaviors. Adult bone marrow progenitor cells (AMPCs) constitute a multipotent heterogeneous population. We hypothesized that microtopographies could direct AMPCs lineage-specific differentiation. AMPCs isolated from Sprague-Dawley rats were CD45 depleted, expanded, and plated at 10(5) cells/cm2 on epoxy-microfabricated: (1) 60-microm-deep grooves with 95-microm pitch (D60P95), (2) 55-microm-wide and 10-microm-deep squares (W55D10), (3) 30-microm-deep grooves with 45-microm pitch (D30P45), (4) 17-microm-wide and 10-microm-deep pillars (W17D10), and (5) smooth control. AMPCs were cultured using expansion, chondrogenesis, or osteogenesis supporting media. Cell cultures were examined by scanning electron microscopy, qRT-PCR, and immunostaining at 2, 9, 16, and 23 days after plating. Expressions of osteogenesis-related genes, such as Runx-2, alkaline phosphatase, osteopontin, osteocalcin, and parathyroid hormone-related protein receptor (PTHr), and chondrogenesis-associated genes, such as Sox-9, type II collagen, and aggrecan, were determined. In expansion medium, W55D10 induced a transient increase of Sox9 expression. Compared with smooth surfaces, type II collagen mRNA and protein expressions in chondrogenic medium were significantly upregulated on W55D10 by day 23. In contrast, osteocalcin and PTHr expressions were significantly increased on D30P45 in osteogenic medium. We have demonstrated that W55D10 and D30P45 enhanced AMPCs chondrogenic and osteogenic terminal differentiation with appropriate culture conditions.

Regulation of epithelial cell growth factor receptor protein and gene expression using a rat periodontitis model.

BACKGROUND AND OBJECTIVE: Regulation of epithelial cell behavior associated with periodontitis is not well elucidated but many responses will ultimately be regulated by growth factor receptors. Using a rat experimental periodontitis model, protein and gene expression of select growth factor receptors in junctional and pocket epithelium were examined. MATERIAL AND METHODS: Periodontal disease was induced by daily topical application of lipopolysaccharide using an established protocol. Animals were killed at time 0 (control), and at 2 and 8 wk. Frozen tissue samples were collected from the right palatal gingival soft tissue, and the left periodontal tissues were decalcified and embedded in paraffin. Laser microdissection and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify keratinocyte growth factor receptor (KGFR), hepatocyte growth factor receptor (HGFR), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) gene expression, and in situ RT-PCR localized these increases to specific epithelial cells. Receptor protein expression was examined immunohistochemically. In cell culture, induction of HGFR and KGFR protein expression by serum, lipopolysaccharide and pro-inflammatory cytokines were examined using flow cytometry. RESULTS: Eight-week tissue samples exhibited histological changes consistent with periodontitis. KGFR and HGFR gene and protein expression were significantly induced at the 8 wk time point. KGFR expression was significantly up-regulated in basal and parabasal pocket epithelial cells, but HGFR was up-regulated throughout the pocket epithelium. In cell culture serum, lipopolysaccharide and pro-inflammatory cytokines, interleukin-1beta and tumour necrosis factor-alpha significantly induced KGFR protein receptor expression, but HGFR expression was only induced by serum. CONCLUSION: KGFR and HGFR are highly up-regulated in this model of periodontal disease and may play a significant role in regulating the proliferation and migration of pocket epithelium.

Successful periodontal maintenance of a case with Papillon-Lefèvre syndrome: 12-year follow-up and review of the literature.

BACKGROUND: Various approaches to treating the periodontal condition associated with Papillon-Lefèvre syndrome have been reported. These include oral hygiene instruction, use of mouthrinses, frequent debridement, multiple antibiotic regimens, periodontal surgery, extraction of hopeless teeth, and extraction of all deciduous teeth. Because Papillon-Lefèvre syndrome is rare, most publications are case reports, and very few document long-term successful treatment of the periodontal condition. METHODS: In 1986, a 3.5-year-old Indo-Canadian male was diagnosed with Papillon-Lefèvre syndrome and began periodontal treatment. Initial therapy consisted of debridement every 3 weeks, a 0.12% chlorhexidine mouthrinse, 2 regimens of metronidazole, and oral hygiene instruction for his parents. After 10 months it became apparent that the treatment was having little beneficial effect, since the periodontal destruction continued and teeth 51 and 61 exfoliated. At age 4, all remaining deciduous teeth were extracted and complete dentures inserted for the following 2-year edentulous period; then a 3-month maintenance schedule was maintained. RESULTS: The patient is now 17 years old and all his adult teeth are present with the exception of the third molars. His oral hygiene varies between moderate and good, with his most recent plaque score at 80% effectiveness. There are no probing depths greater than 4 mm, with the exception of the distal of the lower second molars where opercula are present. CONCLUSIONS: Extraction of all the deciduous teeth followed by a period of edentulousness may partially explain the fact that there has been no recurrent attachment loss in the permanent teeth up to age 17. Other explanations are discussed as part of the literature review of Papillon-Lefèvre syndrome.

Naturally occurring periodontal bone loss in the wild deer mouse, genus Peromyscus.

BACKGROUND: Historically, animal models for the study of periodontal diseases have incorporated surgically created defects, plaque retentive ligatures, as well as soft and high-sucrose diets which may not accurately reflect progression of the natural disease. Spontaneous periodontal disease is seen in a few animal species, but these are often expensive to maintain and are unsuitable for manipulation using advanced molecular biology techniques. Mice are inexpensive, easy to maintain, and are routinely used for transgenic experiments and are therefore an optimal animal for research purposes. However, it is commonly accepted that mice do not spontaneously develop periodontal disease. The purpose of this study was to determine if a mouse population that exhibits periodontal breakdown in the wild could be found, allowing for genetic manipulation of naturally occurring periodontal disease. METHODS: We examined over 2,500 dry skulls of several Peromyscus species from various locations and habitats on the west coast of North America for periodontal bone loss in the molars, using furcation involvement as an indicator of disease severity. Alveolar bone loss was classified as Grade I) horizontal component of bone loss in the furcations; II) through-and-through furcations; and III) through-and-through furcations with alveolar bone loss into the apical third of the root. RESULTS: The proportions of individual mice experiencing bone loss were 3.8% for Class I-III involvement, 1.3% for Class II-III involvement, and 0.5% for Class III alone. Three subspecies of P. keeni and one subspecies of P. maniculatus had periodontal disease prevalences in 7% to 13.5% of their samples. Mice from isolated islands had 1.8- to 4.7-fold higher disease prevalence than those located on the mainland, with even greater prevalence on small islands. No statistically significant differences between genders were found. CONCLUSIONS: It appears that periodontal disease is far more common in this mouse genus than previously believed. Some of the subspecies demonstrated severe periodontal disease at a prevalence comparable to that found in humans.

Dental unit waterline contamination and its possible implications during periodontal surgery.

BACKGROUND: Dental unit waterline contamination has become a concern to clinical dentistry. This concern arises from the fact that bacteria sloughed from established biofilms in dental unit waterlines increase heterotrophic bacteria counts in water exiting these units. METHODS: Scanning microscopy and bacterial viability staining were used to examine the sessile and planktonic biofilm present in dental unit waterlines and water samples, respectively. In addition, the limulus amebocyte assay was used to measure the lipopolysaccharide (LPS) levels in water samples. RESULTS: All dental unit waterlines were coated with a well-established biofilm made up of filamentous and bacillus-like microorganisms. Water samples collected from these dental units contained high numbers of individual bacteria and bacterial aggregates. A viability staining technique identified significantly more bacteria in water than could be cultured, and 64% of the total bacterial population stained as nonvital. Since the bacterial load (viable and nonviable) was high, we examined the LPS in dental unit water samples. The mean LPS levels in water collected from high-speed and air/water lines in use were 480 and 1,008 endotoxin units (EU)/ml. This was significantly higher than the mean level of 66 EU/ml found in water samples collected from adjacent clinic sinks. The LPS level at the start of the day (2,560 EU/ml) was reduced by 70% with 1 minute of flushing (800 EU/ml). Flushing times of 5 and 10 minutes were not able to reduce LPS levels to zero. CONCLUSION: The presence of high heterotrophic bacterial counts, sloughing biofilm, and high LPS levels are discussed in relation to patient risk and periodontal wound healing biology.

Exogenous phospholipase C stimulates epithelial cell migration and integrin expression in vitro.

Phospholipase C secreted by bacterial pathogens has been identified as a virulence factor in several human diseases and has been implicated in impeding wound healing. The role of phospholipase C in the intracellular signal control of epithelial growth was studied in normal human skin keratinocytes cultured in conditions simulating aspects of wound healing. Bacillus cereus phospholipase C decreased cell-cell contact and increased cell migration resulting in disruption of the advancing epithelial sheet. Phospholipase C-induced migration was blocked by inhibitor of the phosphoinositol signal transduction pathway neomycin sulfate and protein kinase C inhibitor RO-31-8220. Induced migration was associated with elevated levels of matrix metalloproteinase-9 which, when blocked by tissue inhibitor of metalloproteinase-1, was accompanied by a loss of migration. Adhesion studies showed that phospholipase C treatment enhanced cell binding to fibronectin, vitronectin and collagen IV. Immunostained phospholipase C-stimulated cells cultured on fibronectin showed enhanced expression and relocation of the integrin subunits alpha(v), alpha5 and beta1. Confocal microscopy showed that phospholipase C-induced levels of integrin subunit beta1 were predominantly deposited on the basal surface of the cell apparently in focal contacts and associated with actin stress fibers. These results indicate that exogenous phospholipase C signaling from a bacterial source may play an important role in perturbing normal reepithelialization via altered expression of integrins and matrix metalloproteinase-9.

An evaluation of sampling and laboratory procedures for determination of heterotrophic plate counts in dental unit waterlines.

BACKGROUND: The high numbers of heterotrophic microorganisms that have been cultured from dental unit waterlines (DUWs) have raised concern that this water may exceed suggested limits for heterotrophic plate counts (HPCs). The main purpose of this investigation was to examine HPC variability in DUWs and to examine in detail the effect of laboratory processing of water samples on HPC values. METHODS: Water samples were collected from dental offices either at the beginning of or during the clinic day and were transported to the laboratory, where they were analyzed. RESULTS: Measuring HPC levels within an office would involve testing all units, because significant differences were found between units connected to the same municipal water supply. Within a unit, the average microbial count from high-speed lines was approximately twice the average count from air/water lines. The laboratory processing of water samples significantly affected the numbers of heterotrophic microorganisms that were recovered. Incubation temperature, time and media, as well as neutralization of residual chlorine, all had significant effects on the HPC values. However, no significant differences in microbial counts were found between samples plated with the spread plate method on R2A agar and those plated with the pour plate method with Plate Count Agar. CONCLUSIONS: Dental organizations have suggested target limits in terms of numbers of heterotrophic microorganisms recovered in water from dental units, but standards for laboratory handling must be established as well. A protocol for sample collection and laboratory handling is proposed.

The periodontal disease classification system of the American Academy of Periodontology--an update.

Until recently, the accepted standard for the classification of periodontal diseases was the one agreed upon at the 1989 World Workshop in Clinical Periodontics. This classification system, however, had its weaknesses. In particular, some criteria for diagnosis were unclear, disease categories overlapped, and patients did not always fit into any one category. Also, too much emphasis was placed on the age of disease onset and rate of progression, which are often difficult to determine. Finally, no classification for diseases limited to the gingiva existed. In 1999, an International Workshop for a Classification of Periodontal Diseases and Conditions was organized by the American Academy of Periodontology to address these concerns and to revise the classification system. The workshop proceedings have been published in the Annals of Periodontology. The major changes to the 1989 proceedings and the rationale for these changes are summarized here. In addition, the potential impact of these changes is discussed.

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin.

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.

Collagenase-3 (matrix metalloproteinase-13) expression is induced in oral mucosal epithelium during chronic inflammation.

Increased proliferation of mucosal epithelium during inflammation is associated with degradation of subepithelial connective tissue matrix and local invasion of the epithelial cells. Here we have studied, whether collagenase-3 (MMP-13), a collagenolytic matrix metalloproteinase with an exceptionally wide substrate specificity, is expressed in the epithelium of chronically inflamed mucosa. Examination of human gingival tissue sections from subjects with chronic adult periodontitis with in situ hybridization revealed marked expression of MMP-13 in basal cells of some epithelial rete ridges expanding into connective tissue. Immunohistochemical staining demonstrated that these cells also expressed strongly laminin-5, suggesting that they are actively migrating cells. A strong signal for MMP-13 mRNA was occasionally also noted in the suprabasal epithelial cells facing the gingival pocket, whereas no collagenase-1 (MMP-1) mRNA was detected in any areas of the epithelium. MMP-13 expression was also detected in fibroblast-like cells associated with collagen fibers of the inflamed subepithelial connective tissue. In organ culture of human oral mucosa, MMP-13 mRNA expression was observed in epithelial cells growing into connective tissue of the specimens. Regulation of MMP-13 expression was examined in cultured normal nonkeratinizing epithelial cells isolated from porcine periodontal ligament. In these cells, MMP-13 expression at the mRNA and protein level was potently enhanced (up to sixfold) by tumor necrosis factor-alpha, transforming growth factor-beta(1), and transforming growth factor-alpha and by keratinocyte growth factor in the presence of heparin. In addition, plating periodontal ligament epithelial cells on type I collagen stimulated MMP-13 expression (sevenfold) as compared with cells grown on tissue culture plastic. The results of this study show, that expression of MMP-13 is specifically induced in undifferentiated epithelial cells during chronic inflammation due to exposure to cytokines and collagen. Thus, it is likely that MMP-13 expression is instrumental in the subepithelial collagenolysis and local invasion of the activated mucosal epithelium into the connective tissue.

Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells.

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.

Chymotrypsin-like enzyme secretion is stimulated in cultured epithelial cells during proliferation and in response to Actinobacillus actinomycetemcomitans.

A chymotrypsin-like enzyme was partially purified from culture medium of epithelial cells of human skin, human gingiva and porcine periodontal ligament by aprotinin-affinity chromatography. The enzyme levels from all three cell types were low in quiescent cultures but increased markedly when the cells were allowed to proliferate. The biphasic elution profile of the enzyme from the affinity column closely matched that of alpha-chymotrypsin and the protein comigrated with it on polyacrylamide gels at 27,000 ML. Synthetic substrate tests of purified fractions showed strong chymotrypsin-like but no trypsin-like or elastase-like activity. Inhibition of protease activity and pH optimum in the range of 7.5-8.0 were consistent with chymotrypsin-like enzymes. Secreted activity was found to be significantly increased by phorbol myristate acetate treatment in a time-course that differed from that of elastase-like activity. Keratinocyte growth factor and epidermal growth factor but not transforming growth factor-beta increased the chymotrypsin-like activity in a concentration-dependent manner. The enzyme secretion by epithelial cells was strongly elevated by exposure to 5 of 6 Actinobacillus actinomycetemcomitans strains isolated from plaque samples of juvenile periodontitis patients. These results indicate that chymotrypsin-like enzymes are secreted by proliferative phenotypes of normal epithelial cells. This enzyme may, therefore, play a role in epithelial physiology and in cell response to certain pathogenic bacteria.

Stimulation of collagenase (matrix metalloproteinase-1) synthesis in histiotypic epithelial cell culture by heparin is enhanced by keratinocyte growth factor.

The role of heparin and heparan sulfate in the control of epithelial collagenase production was investigated utilizing a histiotypic cell culture model. The effect of keratinocyte growth factor (KGF), a heparin-binding growth factor, on collagenase secretion was also examined. Heparin, and, to a lesser extent, heparan sulfate induced release of a 58-kDa, gelatin-degrading enzyme which was subsequently identified as the collagenase, matrix metalloproteinase-1. The increase in collagenase secretion by heparin was further enhanced by the addition of KGF. KGF alone did not have any effect. Analysis of secreted radiolabelled proteins showed that the increase in collagenase activity was not due to a general increase in protein synthesis. Synthesis of collagenase protein was specifically increased by heparin and further increased by KGF plus heparin. Heparin and heparan sulfate in combination with KGF may thus have important roles in the regulation of epithelial cell collagenase under conditions such as inflammation and wound healing.

Keratinocyte growth factor stimulation of gelatinase (matrix metalloproteinase-9) and plasminogen activator in histiotypic epithelial cell culture.

The purpose of this investigation was to examine the role that keratinocyte growth factor (KGF) plays in the control of matrix-degrading protease activity in epithelial cells. The culture conditions had a significant effect on cellular responses to the growth factor. In histiotypic culture on porous-polycarbonate membranes, porcine periodontal ligament epithelial cells responded to KGF with increased 92-kDa gelatinase (matrix metalloproteinase [MMP]-9) activity. No such response was observed in cells maintained on plastic plates. Epidermal growth factor and platelet-derived growth factor also increased MMP-9 activity in the histiotypic cultures of epithelial cells. Addition of heparin with KGF produced a further increase in MMP-9 activity, with heparin alone having no effect. Precoating of polycarbonate membranes with matrix components showed that fibronectin and an engineered poly-RGD molecule substrate were required for KGF plus heparin to increase MMP-9 activity. Precoating plastic culture plates with the same proteins did not generate the same response. Concomitant with gelatinase activity, KGF also increased urokinase-type plasminogen activator in the epithelial cells. Thus, KGF appears to be an important regulator of protease secretion in epithelial cells.

Antigenic relationships among oral Actinomyces isolates, Actinomyces naeslundii genospecies 1 and 2, Actinomyces howellii, Actinomyces denticolens, and Actinomyces slackii.

Antigenic relatedness among human strains of oral Actinomyces and similar isolates from cattle has been analyzed by agglutination and immunoblotting. Whole cell agglutination placed A. viscosus serotype II, A. naeslundii serotypes II and III, Actinomyces NV, and strains from numerical taxomonic clusters C1, C2, C3, C4, and C6 into a single group. A. viscosus serotype I cross-reacted weakly with this group. A naeslundii serotype I strains and the cattle isolates Actinomyces denticolens and Actinomyces howellii were distinct. The agglutination results for A. slackii were equivocal. Immunoblots of cell wall extracts developed with non-absorbed sera showed cross-reactivity (23% to 90% antigenic similarity) among all of the strains tested, including A. israelii. The range of antigenic similarities among the group which included strains of A. viscosus serotype II, the A. naeslundii serotypes, and clusters C1, C2, C3, C4, and C6 was from 39% to 89%. Immunoblotting showed that A. howellii and A. denticolens were between 39% and 72% similar to A. naeslundii and A. viscosus. Absorption of antisera with A. israelii cell walls removed antibodies recognizing antigens common to Actinomyces and made the sera more specific. Immunoblotting with absorbed sera supported the grouping and separation of strains shown by agglutination. In some cases, serotypes could be included into a specific taxonomic cluster. A. naeslundii serotype II and Actinomyces NV most closely resembled cluster C1 strains, and A. naeslundii serotype III resembled cluster C1 strains, and A. naeslundii serotype I and A. viscosus serotype I were included into clusters C5 and C7, respectively. The results support a recent proposal that strains of A. viscosus serotype II, A. naeslundii serotypes II and III, and Actinomyces NV be included into A. naeslundii genospecies 2, that A. naeslundii serotype I should be designated A. naeslundii genospecies 1, and that A. viscosus serotype I should be retained distinct from A. naeslundii, as A. viscosus.

Detection of three-walled infrabony defects by subtraction radiography.

An in vitro investigation was undertaken to establish the sensitivity of subtraction radiography for three-walled infrabony defect detection. After the creation of defects of controlled sizes in the interproximal regions of a dried mandible, subsequent radiographs were taken with the help of an occlusal stent. The radiographs were then digitized, and the images were enhanced by frame summation and averaging to reduce electronic noise. Further standardization was facilitated by the use of a step wedge on each radiograph and a computer program to correct variations in processing and exposure. The results show that defect resolution depended not only on the diameter of the defect but also on the mass of the adjacent bone. The smallest detectable lesion was 0.5 mm in diameter in the interproximal region of the premolars. In addition, a change in depth of 1 mm could be detected at the base of three-walled infrabony defects.