A histochemical study of arylaminopeptidases in hydantoin induced hyperplastic, healthy and inflamed human gingiva

Arylaminopeptidase activity in hydantoin induced hyperplastic, inflamed and healthy human gingiva was studied using various N-L-aminoacyl-2-naphthylamines as substrates. The activity was seen to be located in the basal cell layer of the epithelium in the entire connective tissue, but it was strongest just below the epithelium in all tissues. High enzymic activity was also observed in the inflammatory cells as well as in the capillary walls of hydantoinhyperplastic and inflamed gingiva. Strong enzymic activity was obtained when N-L-alanyl-, N-L-methionyl- and N-L-leacyl-2-naphthylamine were used as substrates. Moderate activity was observed with N-L-arginyl-2-naphthylamine and N-Llysyl-2-naphthylamine in other tissues except in healthy gingiva where the enzymic activity was low or nil. To test the possible involvement of aminopeptidase B in the material the reactions were performed both in the presence and absence of 0.2 M sodium chloride, which specifically activates this enzyme. There was no observable enzymic activity in any slices when N-L-prolyl-2-naphthylamine was used as substrate.
The role of different arylaminopeptidases in connection with gingival hyperplasia caused by diphenylhydantoin and the evident absence of enzymes specific to N-L-prolyl-2-naphthylamine are discussed.     

Effect of a Ca(OH)2 solution and a chlorhexidine based detergent on the microbial activity of human carious teeth

Occlusal carious lesions from human molars, preserved in continuous humidity after extraction, were removed using conventional clinical techniques. Bacteriological samples were taken after rinsing the cavity with water only, after experimentally infecting the cavity and after treating uninfected and infected cavities either with a saturated Ca(OH)₂ solution or with a chlorhexidine based detergent. The samples were cultivated on blood agar plates aerobically and anaerobically. Cavities rinsed with water only showed very sparse bacterial growth. After experimental infection the growth was significant, but decreased radically after treatment with the test materials. In order to describe the effect of the two test materials on the microbial enzyme activity in infected dentin, cryostat sections of 10 μm were prepared from undemineralized carious dentin fragments excavated from freshly extracted human teeth. Conventional histochemical techniques were applied to demonstrate the aminopeptidase activity in the sections using N-L-leucyl-2-naphthylamide as a substrate. The aminopeptidase activity of carious dentin was inhibited totally with the Ca(OH)₂ solution, whereas the chlorhexidine based detergent had no effect on the enzyme activity.     

The content of arginine aminopeptidases,hexosamine, and uronic acid sugars in gingival exudate as affected by short term sugar diets

The specific activity of arginine aminopeptidases (which are properly represented by aminopeptidase B) and the amount of hexosamines, uronic acids, serotonin and histamine was determined in gingival exudates obtained from persons kept for five days on various mild sugar diets (including sucrose, xylitol, fructose-xylitol, fructose-sorbitol, fructose-glucose, and sucrose-maltose). The sugars consumed during reduced oral hygiene did not differ as regards their ability to induce aminopeptidase B-activity. The enzyme activity towards N-L-arginyl-2-naphthylamine was somewhat higher in fructose-sorbitol, glucose-fructose, fructose-xylitol and xylitol groups than in the sucrose and sucrose-maltose groups. The sugars did not lead to any differences in the amount of uronic acids and hexosamines in the exudates. This concerned histamine and serotonin as well.     

Cellular enzyme activity associated with tissue degradation following orthodontic tooth movement in man

Enzyme histochemical techniques were used as markers of macrophage activity and differentiation in the periodontal tissues following orthodontic tooth movement in man. The enzymes studied included lactate dehydrogenase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, acid phosphatase and its tartrate resistant isoenzyme, arylsulfatase, aminopeptidase M and prostaglandin synthetase. Chloroacetyl esterase activity was studied in order to detect possible neutrophilic degrading activity. Intense activities of arylsulfatase and prostaglandin synthetase and a moderate activity of aminopeptidase M were found in cells degrading the hyaline zone. However, no activity of tartrate resistant acid phosphatase was found in these cells. Giant cells in contact with bone surfaces adjacent to the hyaline zone exhibited an intense activity of succinic dehydrogenase, tartrate resistant acid phosphatase and aminopeptidase M. Chloroacetyl esterase activity did not change following orthodontic treatment. The results indicate that macrophages in various stages of differentiation were responsible for the degradation of the hyaline zone and alveolar bone during orthodontic tooth movement. The enzymatic differences were probably due to the influence of the immediate cellular environment. Prostaglandin synthetase activity, which may be interpreted as a sign of prostaglandin secretion, was associated with the degradation of the hyaline zone in man.     

Cellular enzyme activity associated with tissue degradation following orthodontic tooth movement in man

Abstract – Enzyme histochemical techniques were used as markers of macrophage activity and differentiation in the periodontal tissues following orthodontic tooth movement in man. The enzymes studies included lactate dehydrogenase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, acid phosphatase and its tartrate resistant isoenzyme, arylsulfatase, aminopeptidase M and prostaglandin synthetase. Chloroacetyl esterase activity was studied in order to detect possible neutrophilic degrading activity. Intense activities of arylsulfatase and prostaglandin synthetase and a moderate activity of aminopeptidase M were found in cells degrading the hyaline zone. However, no activity of tartrate resistant acid phosphatase was found in these cells. Giant cells in contact with bone surfaces adjacent to the hyaline zone exhibited an intense activity of succinic dehydrogenase, tartrate resistant acid phosphatase and aminopeptidase M. Chloroacetyl esterase activity did not change following orthodontic treatment. The results indicate that macrophages in various stages of differentiation were responsible for the degradation of the hyaline zone and alveolar bone during orthodontic tooth movement. The enzymatic differences were probably due to the influence of the immediate cellular environment. Prostaglandin synthetase activity, which may be interpreted as a sign of prostaglandin secretion, was associated with the degradation of the hyaline zone in man.     

Effect of a Ca(OH)2 solution and a chlorhexidine based detergent on the microbial activity of human carious teeth.

Occlusar carious lesions from human molars, preserved in continuous humidity after extraction, were removed using conventional clinical techniques. Bacteriological samples were taken after rinsing the cavity with water only, after experimentally infecting the cavity and after treating uninfected cavities either with a saturated Ca(OH)2 solution or with a chlorhexidine based detergent. The samples were cultivated on blood agar plates aerobically and anaerobically. Cavities rinsed with water only showed very sparce bacterial growth. After experimental infection the growth was significant, but decreased radically after treatment with the test materials. In order to describe the effect of the two test materials on the microbial enzyme activity in infected dentin, cryostat sections of 10 micrometer were prepared from undermineralized carious dentin fragments excavated from freshly extracted human teeth. Conventional histochemical techniques were applied to demonstrate the aminopeptidase activity in the sections using N-L-leucyl-2-naphthylamide as a substrate. The aminopeptidase activity of carious dentin was inhibited totally with the Ca(OH)2 solution, whereas the chlorhexidine based detergent had no effect on the enzyme activity.     

Extractions in kidney transplant recipients: A prospective observational pilot study

BackgroundThe authors undertook a prospective study to determine whether kidney transplant recipients had an increased risk of developing complications, such as local acute infection, alveolitis, increased bleeding, pain, and delayed healing, after tooth extraction.MethodsThe authors selected patients who underwent kidney transplants more than 6 months ago (study group) and patients who had not (control group) older than 18 years who needed to undergo extraction of erupted teeth. The same oral surgeon performed all tooth extractions while the patients were under local anesthesia. Another blind researcher examined the patients 3, 7, and 21 days after tooth extraction. The first end point was occurrence of complications (local acute infection, alveolitis, increased bleeding), and the second end point was socket reepithelialization on day 21.ResultsForty-five tooth extractions were performed on 38 study group participants and 61 on 57 control group participants. There was no statistical difference between the groups regarding the incidence of any complication or delayed socket epithelialization.ConclusionsThe results of this pilot study suggest that there is no difference in postoperative healing after tooth extractions between stable kidney transplant patients and control patients.Practical ImplicationsThis is the first prospective study assessing the frequency of postoperative complications after tooth extraction in kidney transplant recipients.This clinical trial was registered at ClinicalTrials.gov. The registration number is NCT02547753.     

SDF-1在拔牙创软组织愈合过程中的表达及分布

目的:观察拔牙创软组织内基质细胞衍生因子l(SDF-1)的表达及分布,为促进拔牙创愈合提供新思路。方法:Wistar雄性大鼠30只,随机分为10组,每组3只,分别拔除左侧下颌第一磨牙。于拔牙后1、2、4、7、10 d分别采用免疫组织化学染色和RT-PCR技术观察SDF-1在创缘软组织内的分布及表达变化情况。采用SPSS12.0软件包对数据进行统计学分析。结果:免疫组织化学染色显示,拔牙早期创口周围牙龈组织内SDF-1表达增强,染色部位主要分布于牙龈上皮的棘层及基底层细胞胞质与细胞间。越靠近基底层细胞,染色越明显。拔牙后4 d,阳性染色部位主要位于基底细胞层,新生肉芽组织内可见新生血管的内皮细胞和增殖活跃的成纤维细胞阳性表达。拔牙后7 d,牙龈上皮细胞层染色变得均匀,固有层可见少量阳性染色的新生血管内皮细胞及成纤维细胞;拔牙后10 d,牙龈上皮染色特点基本与正常牙龈相似。实时定量PCR检测结果表明,SDF-1mRNA的表达水平在拔牙后1 d达到最高（P<0.01）,第2天开始下降(P<0.05),拔牙后4 d出现第2次峰值（P<0.01）,7 d后其表达水平仍高于对照组(P<0.05),10 d时恢复正常水平(P>0.05)。结论:SDF-1参与拔牙创软组织的早期愈合过程,可能对拔牙创的愈合起着一定的促进作用。     

Partial purification and characterization of arylamidases from human palatine secretions

The secretions (HPS) contained an arylamidase-like enzyme discovered by chromatography on Sephadex G-100 Superfine columns using N-L-alanyl-2-naphthylamine (2NA) as substrate. The enzyme was fractionated in the void volume, suggesting that its molecular weight was 150,000 or higher. It hydrolysed, with decreasing rates, the 2NA of L-alanine, L-leucine, L-methionine and L-phenylalanine, the pH optimum for the best substrate (ala-2NA) being 8.0, alpha-Benzoyl-DL-arginine-2NA was not hydrolysed. p-Chloromercuribenzoate, EDTA, Ca2+ and Zn2+ were inhibitory, whereas chemical modification with typical tyrosyl group reagents did not significantly inactivate the enzyme. Treatment of HPS with Triton X-100 revealed two further arylamidase-like enzymes with lower mol. wt (90,000 and 40,000, respectively). Inhibition characteristics and Cl- effects suggest that one of these enzymes resembles aminopeptidase B (EC 3.4.11.6). HPS also contains endopeptidase activity over a wide pH range (6-9). The number of enzymes in HPS is thus small and most of the peptidolytic activity of HPS in vitro is due to one major enzyme with arylamidase activity.     

Downregulated gene expression of TGF-βs in diabetic oral wound healing

BackgroundHealing of tooth extraction sockets in poorly controlled diabetic patients is often delayed and accompanied by severe infection. The exact cellular and molecular mechanisms underlying the pathogenesis of this complication are still not fully understood.ObjectivesThe purpose of this study was to investigate molecular changes associated with delayed oral wound healing in diabetes.Materials and methodsSix to eight weeks old male type 2 diabetes and age matched control inbred mice were used and maxillary molar tooth extractions were performed. At 4 and 7 days after tooth extraction, the edentulous mucosa of the mice were harvested, and analyzed for histology and gene expression of key wound healing factors.ResultsIn the diabetic model, histological analysis showed that epithelial tissue migration for wound closure was delayed after tooth extraction compared to the control. Quantitative real-time PCR revealed that expression of the TGF-β1, TGF-β2, TGF-β3, TGFβRII and TGFβRIII genes was significantly downregulated in the diabetic model at 4 and 7 days after tooth extraction.ConclusionThese results suggest that delayed wound healing of oral mucosa in diabetes may be associated with decreased expression levels of these regulatory genes which play important roles in controlling epithelial wound closure.     

Aminopeptidase activity in strains of oral streptococci

Ten strains of oral streptococci were analyzed for aminopeptidase activity by isoelectric focusing and crossed immunoelectrophoresis together with enzyme staining procedures. Substrate specificities were determined using 15 aminoacyl-β-naphthylamides. All 10 strains tested showed enzyme activity with the arginine and leucine substrates. None of the strains exhibited activity with the glycine, cystine, or proline substrates. Some strains had multiple enzyme bands with some of the substrates. All strains had aminopeptidase bands located in the pi range 4.1–4.3, except Streptococcus mutans IB and Streptococcus milleri NCTC 10708. The isoelectric profiles of these strains, having enzyme bands in the range 4.45–4.9, differed markedly from the others. These were also the only strains where no arginine aminopeptidase cross-reactivity occurred with the Streptococcus mitis ATCC 903 antiserum.     

Alveolar bone remodeling around immediate implants placed in accordance with the extraction socket classification: a three-dimensional microcomputed tomography analysis

Background: Previous studies assessed bone remodeling after a single tooth extraction; however, the effect of multiple contiguous teeth extractions around immediate implant remains unknown. The aim of this microcomputed tomographic investigation is to analyze the alveolar bone remodeling around immediate implants placed in accordance with the extraction socket classification (ESC). Methods: Under general anesthesia, 10 beagle dogs underwent atraumatic tooth extractions. Animals were randomly divided into three groups, with 16 sites per group: 1) ESC‐1, single tooth extraction; 2) ESC‐2, two contiguous teeth extraction; and 3) ESC‐3, more than two contiguous teeth extractions. Immediate implants were inserted in each socket, and postoperative plaque control measures were undertaken. After euthanasia, the jaw segments were evaluated for bone thickness, marginal bone loss (MBL), and bone‐to‐implant contact (BIC) using microcomputed tomography. Results: The mean buccal bone thickness (P <0.05) and MBL (P <0.05) was compromised in jaws in ESC‐3 compared to those in ESC‐1 and ESC‐2. The BIC was significantly higher among jaws in ESC‐1 compared to those in ESC‐2 and ESC‐3 (P <0.05). There was no significant difference in the buccal bone thickness, MBL, and BIC among the groups in the maxilla and mandible. Lingual bone remodeling did not reveal any significant differences among the groups in either jaw. Conclusion: Buccal bone remodeling is significantly more extensive around immediate implants placed in multiple contiguous tooth extraction sites compared to immediate implants placed in single tooth extraction sites.     

Influence of trans-operative complications on socket healing following dental extractions

AIM: Extraction healing complications have been attributed to several factors. The influence of trans-operative complications on an extraction site wound healing was the focus of this investigation. METHODS AND MATERIALS: This prospective study was conducted at the Oral Surgery Clinic of the Department of Oral and Maxillofacial Surgery of the Lagos University Teaching Hospital (LUTH) in Nigeria . Subjects selected were those referred for one or two adjacent extractions and who satisfied the inclusion criteria for the study. The relevant pre-operative information recorded for each patient were age and sex of patient, indications for extraction, time taken to extract the tooth, tooth/teeth removed, and any trans-operative complications. Extractions were performed with dental forceps, elevators, or both under local anaesthesia. Patients were blindly evaluated on the third and seventh post-operative day for socket healing assessment without reference to pre-operative information on the patients. RESULTS: Seventy-three (24.25%) of 301 teeth considered for socket healing assessment had various trans-operative complications due to accidental crown, root, or alveolar bone fractures. Of the 73 extractions with trans-operative complications during extraction, 18 developed a socket healing complication, while 17 of the 228 extractions without trans-operative complications developed socket healing complications (p = .000). The mean (SD) time taken to extract teeth developing healing complications was also found to be significantly longer than those without healing complications (p < .01). CONCLUSIONS: The study demonstrated the combination of tooth/bone fragments in the socket and increased time of extraction due to trans-operative complications and accidents predispose to the development of extraction site wound healing disturbance.     

Vertical and horizontal ridge alterations after tooth extraction in the dog: flap vs. flapless surgery

Objective: To compare ridge alterations after flap and flapless tooth extraction in the vertical and horizontal dimension in the dog model. Material and methods: This study was carried out on five Beagle dogs. Four extractions were performed in the lower jaw of each dog (two per side. Pm3, Pm4). At the time of tooth extraction, flap surgery was performed on one side (control group). On the contra‐lateral side, a flapless extraction was performed (test group). Mesial sockets were left untreated on both sides. After 3 months of healing, the dogs were sacrificed and prepared for histological analysis. Results: Ten samples were evaluated on each group. The vertical difference in height between the buccal and lingual crest was 1.48 mm for the flap, and 1.22 mm for the flapless group. The horizontal dimension of the ridge was 4.41 mm (at 1 mm from the crest), 5.72 mm (at 3 mm from the crest) and 6.67 mm (at 5 mm form the crest) in the flap group. In the flapless group, the measurements were 4.5, 5.58 mm and 6.44 at 1, 3 and 5 mm from the crest, respectively. Conclusion: Evaluating ridge alterations in the vertical and horizontal dimension after 3 months of healing following tooth extraction, results for the flap and the flapless group were very similar. To cite this article:
Blanco J, Mareque S, Liñares A, Muñoz F. Vertical and horizontal ridge alterations after tooth extraction in the dog: flap vs. flapless surgery.
Clin. Oral Impl. Res. 22 , 2011; 1255–1258.
doi: 10.1111/j.1600‐0501.2010.02097.x     

Histochemical demonstration of carbonic anhydrase activity in the odontogenic cells of the rat incisor

Aldehyde-fixed, EDTA-demineralized frozen sections of the rat maxillary incisor were histochemically stained for carbonic anhydrase activity, by use of Hansson's method. Intense staining was observed in the odontoblasts, all types of epithelial cells of enamel organ in the maturation zone, cementoblasts, and the cells of the lingual dental sac. Less intense but consistent staining was observed in all types of epithelial cells of odontogenic origin directly facing the pulp and pulp cells adjacent to the odontoblast cell layer in the apical part of the pulp, and was considered due to the carbonic anhydrase-catalyzed reaction. Staining of these cells was completely inhibited by heat pretreatment (120 degrees C, 30 min), 10(-6) mol/L acetazolamide in the incubation medium, incubation by continuous immersion under the liquid surface, and omission of the substrate, NaHCO3. The dentin also exhibited heavy staining which was inhibited by the heat pre-treatment. However, this dentinal staining resisted the inhibition by 10(-3) mol/L acetazolamide and was not inhibited by incubation by continuous immersion or incubation without the substrate NaHCO3. The dentinal staining was thus judged to have been due to non-enzymatic cobalt precipitation.     

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??Objective    To study the mechanism of periodontal tissue repair of transplanted tooth by analyzing the imaging and histological changes of autogenous transplanted tooth in Beagle dog. Methods    For a 8-months-old beagle??the left mandibular premolar region was used as receptor region for autologous tooth transplantation of anterior teeth. X-ray examination was done after operation to observe the periodontal healing of transplanted teeth. Results    X-ray at one month after operation showed that the No.1 tooth ??left mandibular second premolar?? has a narrowed periodontal gap??a small periapical radiolucency and external resorption at tooth neck; No. 2 tooth ??the left mandibular first premolar?? has a internal resorption in the upper 1/3 root canal. Two-month post-operative X-ray examination indicated the periodontal healing of transplanted teeth was perfect. The external resorption of No. 1 tooth enlarged and internal resorption of No. 2 tooth remained the same. HE staining results showed that new fibrous bundle was formed at the root tip. A large number of lymphocytes and some plasmocytes exited at the tooth neck??pulp cavity and root tip. Conclusion    Autogenous tooth transplantation can achieve good periodontal membrane healing. It is important to reduce the mechanical damage to tooth neck while pulling out the teeth. The timing of the dental pulp treatment after transplantation is one of the key elements to reduce postoperative complications.     

Proteolytic profile of Treponema vincentii ATCC 35580 with special reference to collagenolytic and arginine aminopeptidase activity

The proteolytic profile of Treponema vincentii ATCC 35580 was studied using PZ-PLGPA (phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-argi-nine; a substrate of bacterial collagenases) and amino acid 2-naphthylamides (2NA) as substrates. The cell extracts showed high activity toward PZ-PLGPA, Na -L-arginyl-2NA and N γ-L-glutamyl-2NA. Gel permeation chromatography revealed 2 major endopeptidases (I and II) hydrolyzing PZ-PLGPA, the molecular weights of which were 75,000 and 23,000, respectively. Enzyme I was stable enough for subsequent fast protein liquid chromatography on an anion exchange column. The enzyme had a broad pH optimum of 6.5 to 7.5 with PZ-PLGPA as substrate, hydrolyzed gelatin and was moderately inhibited by metal chelators, but was very sensitive to p -chloromercuribenzoic acid (PCMB). Enzyme II with a pH optimum of 7 to 8 was more labile, quite sensitive to PCMB and moderately inhibited by chelators. A high-molecular weight arginine aminopeptidase (mol. wt. > 200,000) was sensitive to PCMB and showed a value of 0.55 mM for K_m in the hydrolysis of Na -L-arginyl-2NA. The hydrolysis of PZ-PLGPA and gelatin suggests that this organism may contain collagenolytic proteinases. Because the insoluble proteinase substrate Azocoll was not hydrolyzed, these enzymes may be active on soluble collagenous substances only. T. vincentii ATCC 35580 typifies an organism rich in PZ-PLGPA-endopeptidase, arginine aminopeptidase and γ-glutamylpeptidase activity.     

Bone healing after dental extractions in irradiated patients: a pilot study on a novel technique for volume assessment of healing tooth sockets

The aim of this study was to evaluate longitudinally the bone-healing process by measuring volumetric changes of the extraction sockets in head and neck cancer patients undergoing radiotherapy after tooth extraction. A total group of 15 patients (nine males, six females) undergoing tooth extraction at the Department of Periodontology (University Hospital KULeuven) were enrolled after giving informed consent. In seven patients, teeth presenting a risk for complications and eventual radionecrosis were extracted prior to the radiotherapeutical procedure. Monitoring of bone healing was performed by evaluating the volumetric changes of the alveoli by cone beam CT scanning (CBCT) at extraction and after 3 and 6 months. In parallel, a similar longitudinal evaluation of extraction sites was done in a control group of eight patients. Within this pilot-study, a total of 15 healing extraction sockets were evaluated and followed up. There was a significant difference in volumetric fill up of extraction sockets in test group vs. control group at three (37.1 ± 7.9%) vs. (54.6 ± 4.0%) and 6 months (47.2 ± 8.8%) vs. (70.0 ± 7.3%), respectively. The present pilot study demonstrated the clinical usefulness of CBCT for evaluation of extraction socket healing. The study objectively demonstrates the delayed bone healing after tooth extraction in irradiated head and neck cancer patients. Considering the limitations of this pilot study, a potential effect of radiotherapy on further jaw bone healing after pre-therapeutic tooth extractions should be further explored.     

Studies on the aminopeptidase activities of Porphyromonas gingivalis

Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV).     

An immunohistochemical study of matrix molecules associated with barrier membrane-mediated periodontal wound healing

Guided tissue regeneration (GTR) is a clinical procedure developed to facilitate periodontal regeneration by using barrier membranes to selectively promote the repopulation of a periodontal defect by periodontal ligament and bone cells at the expense of epithelial and gingival connective tissue cells. The aim of this study was to gain insight into the biological events occurring during membrane mediated periodontal wound healing by examining the immunohistochemical expression of a number of extracellular matrix components in tissues treated via the GTR technique. Experimental periodontal defects were created around the second premolar tooth in 4 dogs and wound closure was achieved by application of expanded polytetrafluoroethylene membranes around each tooth and flap positioning coronal to the cementoenamel junction. The dogs were sacrificed after a 4-wk healing period, block dissections of the part of the mandible containing the experimental tooth were obtained and paraffin sections were prepared. Using standard immunohistochemical techniques, the sections were stained with a monoclonal antibody against bone morphogenetic proteins 2 and 4 (BMP-2 and -4) and polyclonal antibodies against collagen I, collagen II, decorin, biglycan, bone sialoprotein, osteopontin and osteocalcin. Collagen I was predominantly localized within the regenerating bone, whereas collagen III staining was more abundant in the soft connective tissues of the defect. Decorin and biglycan staining was faint within the extracellular matrix of the regenerating defect, although both proteoglycans exhibited intense intracellular localization within some of the cells inhabiting the defect. The staining for BMP-2 and -4 was weak within the bone but strong within the extracellular matrix of the regenerating soft tissue. Osteopontin and bone sialoprotein were strongly localized in the regenerating bone and cementum found within the defect. Osteocalcin staining was present in both the regenerating and mature cementum and associated cementoblasts, and it was relatively weaker in the regenerating bone compared to the mature bone. The observed pattern of immunolocalization of the extracellular matrix macromolecules suggests that the heterogeneous cell population filling the GTR wound had created an environment that was conducive to periodontal regeneration.