Novel biological activity of ameloblastin in enamel matrix derivative

Objective

Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells.

Material and Methods

Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Results

Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein.

Conclusion

The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues.     

Effects of enamel matrix proteins on tissue formation along the roots of human teeth

OBJECTIVE: Enamel matrix-derived proteins (EMD) are thought to trigger the formation of acellular extrinsic fibre cementum (AEFC), while other reports indicate that EMD may have osteogenic potential. The aim of the present study was to characterize the tissues developing on the root surface following application of EMD. METHODS: Twelve human periodontitis-affected teeth, scheduled for extraction, were treated with EMD. Two to 6 weeks later, the teeth were extracted, demineralized and processed for embedding in acrylic and epoxy resins. New tissue formation was analysed by light and transmission electron microscopy. RESULTS: New tissue formation on the root was observed in the notch and on both scaled and unscaled root surfaces distant of the notch area in six defects. The newly formed tissues on the root were thick, collagenous, devoid of extrinsic fibres, and had an irregular surface contour. The presence of electron-dense, organic material in the collagenous matrix indicated at least partial mineralization. Embedded cells were numerous and the cells on the matrix surface were very large in size. Abundant rough endoplasmic reticulum and a prominent Golgi complex were evident. The presence of a split between the treated root surfaces and the newly formed tissue was a common observation, as was the presence of bacteria and host cells in the interfacial gap. CONCLUSION: Following treatment with EMD, a bone-like tissue resembling cellular intrinsic fibre cementum may develop on the root surfaces, instead of AEFC. Furthermore, EMD may both induce de novo formation of a mineralized connective tissue on scaled root surfaces and stimulate matrix deposition on old native cementum. Interfacial bonding appeared to be weak after 6 weeks of healing.     

自体牙周膜细胞移植对狗牙周组织再生的影响

目的　对应用自体牙周细胞移植结合e PTFE膜引导的牙周组织再生的动物实验进行评价。方法　将 6只成年狗的 36颗牙分为实验组和对照组 ,每组 18颗牙。在人工制造的牙周缺损中 ,进行体外培养的自体牙周细胞移植结合GTR法为实验组 ,单纯应用GTR法为对照组。 6周后切片行牙周组织学观察。结果　实验组新生牙槽骨、牙周膜组织及牙骨质的修复再生效果明显好于对照组 (P <0 0 5 ) ;实验组牙槽骨再生高度平均为 (4 0 0± 0 13)mm ,对照组为 (3 0 9± 0 2 8)mm。结论应用自体牙周膜细胞移植结合e PTFE膜引导牙周组织再生可促进狗牙周组织的再生     

Periodontal regeneration with enamel matrix derivative in reconstructive periodontal therapy: a systematic review

Background: Enamel matrix derivative (EMD) is commonly used in periodontal therapy. The aim of this systematic review is to give an updated answer to the question of whether the additional use of EMD in periodontal therapy is more effective compared with a control or other regenerative procedures. Methods: A literature search in MEDLINE (PubMed) for the use of EMD in periodontal treatment was performed up to May 2010. The use of EMD in treatment of intrabony defects, furcations, and recessions was evaluated. Only randomized controlled trials with ≥1 year of follow‐up were included. The primary outcome variable for intrabony defects was the change in clinical attachment level (CAL), for furcations the change in horizontal furcation depth, and for recession complete root coverage. Results: After screening, 27 studies (20 for intrabony defects, one for furcation, and six for recession) were eligible for the review. A meta‐analysis was performed for intrabony defects and recession. The treatment of intrabony defects with EMD showed a significant additional gain in CAL of 1.30 mm compared with open‐flap debridement, EDTA, or placebo, but no significant difference compared with resorbable membranes was shown. The use of EMD in combination with a coronally advanced flap compared with a coronally advanced flap alone showed significantly more complete root coverage (odds ratio of 3.5), but compared with a connective tissue graft, the result was not significantly different. The use of EMD in furcations (2.6 ± 1.8 mm) gave significantly more improvement in horizontal defect depth compared with resorbable membranes (1.9 ± 1.4 mm) as shown in one study. Conclusions: In the treatment of intrabony defects, the use of EMD is superior to control treatments but as effective as resorbable membranes. The additional use of EMD with a coronally advanced flap for recession coverage will give superior results compared with a control but is as effective as a connective tissue graft. The use of EMD in furcations will give more reduction in horizontal furcation defect depth compared with resorbable membranes.     

Enamel matrix derivative versus guided tissue regeneration in the presence of nicotine: a histomorphometric study in dogs

AIM: The goal of this histometric study was to compare the healing process of dehiscence-type defects treated by enamel matrix derivative (EMD) or guided tissue regeneration (GTR) under the effect of nicotine in the dog model. MATERIALS AND METHODS: Eight mongrel dogs were used. Buccal osseous dehiscences were surgically created on the mesial roots of the mandibular third and fourth pre-molars. The defects were exposed to plaque accumulation for 3 months. After this period, the defects were randomly assigned to one of the treatments: open flap debridement (OFD), EMD or GTR with a resorbable membrane. During 4 months, the dogs received subcutaneous administration of nicotine (2 mg/kg twice a day with a 12 h interval between the applications). After this period, the animals were killed and the blocks were processed. The histometric parameters evaluated included gingival recession, epithelial length, connective tissue adaptation, new cementum and new bone. RESULTS: A superior length of new cementum was observed in the sites treated by EMD in comparison with OFD (p< or =0.05). No statistically significant differences were observed between GTR and the other groups. CONCLUSIONS: In the presence of nicotine, EMD may promote more new cementum formation than OFD while GTR failed to provide a significant difference.     

Comparison of infrabony defects treated with enamel matrix derivative versus guided tissue regeneration with a nonresorbable membrane

AIM: The purpose of the present multicenter clinical trial was to compare the efficacy of two different procedures in the treatment of infrabony defects: guided tissue regeneration (GTR) with nonresorbable membranes and enamel matrix derivative (EMD). MATERIAL AND METHODS: Six centers participated in this study. Ninety-eight patients with an interproximal infrabony defect were selected. All patients were treated with an initial phase of scaling and root planing, and at the study's baseline the selected defects presented a value of probing depth (PD) > or =6 mm with an infrabony component > or =4 mm. Forty-nine patients were treated with GTR procedures (using ePTFE membranes (Gore-Tex W.L. Gore and Associates, Flagstaff, AZ, USA)) and forty-nine with EMDs (Emdogain (U Biora AB Malm, Sweden)). The efficacy of each treatment modality was investigated through covariance analysis. RESULTS: The patients were reevaluated at one year postop. Probing attachment level (PAL) gain and PD reduction were analyzed. In the Emdogain group the PAL before surgery (PAL 0) and the PD before surgery (PD 0) were respectively 9.9+/-1.4 and 8.5+/-1.6 mm. The PAL gain and the PD reduction at 1 year postsurgery were respectively 4.1+/-1.8 and 5.3+/-1.9 mm. The group of patients treated with membranes showed that PAL 0 and PD 0 were respectively 8.9+/-1.9 and 8.1+/-1.9. The PAL gain was 4.3+/-1.9 mm and the PD reduction was 5.6+/-1.5 mm. The mean PAL gain expressed by percentage (PAL gain/PAL 0) for the group treated with EMD was 41%, while it was 48% for the group treated with GTR. Results from our analysis suggest that there is no statistically significant difference between GTR and EMD treatments in terms of PAL gain, PD reduction and recession variation. Applying the regression model to a group of patients with a PAL 0 > or =8 mm, we observed a better clinical outcome in terms of PAL gain (difference of 0.3 mm) in patients treated with the GTR procedure compared to those treated with EMD. Covariance analysis showed a strong correlation in both groups of patients between PAL gain and full mouth bleeding score, and between PAL gain and defect morphology and depth.     

Collagen gel and membrane in guided tissue regeneration in periodontal fenestration defects in dogs

Abstract The effect of a collagen gel matrix as a submembranous space-maintaining material was evaluated in guided tissue regeneration procedures. In 4 dogs, contralateral surgical circular fenestration defects, 5 mm in diameter, were produced at the midbuccal aspect of the alveolar bone in 8 maxillary canines. Removal of bone, PDL and cementum was complete. Experimental sites were filled with collagen gel and covered with collagen membranes; control sites were covered with collagen membranes and the underlying space was spontaneously filled with blood. Mucogingival flaps were repositioned. Histological and histomorphometric observations, 6 weeks post-surgery, indicated that defects covered by collagen membranes presented the most impressive regeneration with almost complete coverage of the denuded root by new cementum (98.4%) and new bone (63.2%). In the experimental defects. 83.5% coverage of new cementum with only 21.9% new bone regeneration was observed. These results suggest that collagen gel. interfered with healing by PDL and bone-derived cells in the submembranous space.     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

Healing of sites within the dog periodontal ligament after application of cold to the periodontal attachment apparatus

The potential of periodontal ligament-derived tissues to regenerate periodontal attachment after cryosurgical trauma to the PDL in dogs was evaluated. The buccal alveolar plate of each canine tooth was exposed by a semi-lunar excision. A 3 mm thick cryoprobe, cooled to -81 degrees C, was placed on the bone 5 mm apical to the crest for 10 s. This induced cellular devitalization in the bone directly in contact with the probe and the PDL under it. The freezing-thawing cycle was repeated 3 times. Control sites were sham-operated at room temperature. Histologic sections from the center of the lesions were obtained from 1 h, 48 h and 30 d specimens. 1-h control and experimental histologic sections were similar. At 48 h post-surgery, the cellular component of the frozen PDL could not be identified and inflammatory response was minimal. The collagenous framework, however, appeared to form a continuum between the alveolar bone and cementum. Lacunae in the bone at the frozen segment were empty. The injured PDL was surrounded by normal PDL. Control specimens appeared normal. At 30 d, the PDL space in the frozen segments was populated by PDL-like tissue which did not differ significantly from the PDL coronal or apical to it. Collagen fibers appeared to be attached to the cementum on one side and to the alveolar bone on the other. Bone resorption or ankylosis was not observed in the experimental sites. It is suggested that the extracellular matrix in the devitalized area was preserved, supporting regeneration of the cryolesion.     

Attachment, proliferation and differentiation of periodontal ligament cells on various guided tissue regeneration membranes

The purpose of this study was to evaluate the biological effects of guided tissue regeneration (GTR) membrane materials, per se, on the periodontal tissue regeneration. Rat periodontal ligament (PDL)-derived cells were used to study the attachment, proliferation and differentiation, in vitro, on various GTR membranes. Five commercially available membranes bovine type I collagen (BioMend; BM), bovine type I atelocollagen (Tissue Guide; TG), polylactic acid (Epi-Guide; EG), co-polymer of polylactic acid and polyglycolic acid (Resolute; RL) and expanded polytetrafluoroethylene: e-PTFE (Gore Tex; GT)-were examined. A 3 x 3 mm section of the membrane was fixed to the bottom of a 35 x 10 mm style culture dish and plated with 2 ml of cell suspension at an initial density of 5 x 10(4) cells/ml in culture medium with 10% fetal bovine serum. For cell growth analysis, the specimens were fixed with 10% buffered formalin and stained with hematoxylin at 1.5 hours and 1, 3 and 5 days after cell seeding. The number of cells included in a unit area of 0.25 mm2 were counted under light microscopy. As a comparative scaffold of cell proliferation, a plastic cover for cell culture slip (Celldesk; CD) was used. For analysis of cell differentiation, activity of alkaline phosphatase (ALP) and calcification were histochemically revealed after 2-week cultivation. The initial number of PDL cells attached to the membrane at 1.5 hours after cell seeding was different among membranes. RL, TG and EG had the same level of attached cell numbers as that on CD, while the cell numbers on GT and BM were significantly lower than that on CD (p < 0.01). The rate of cell proliferation with time also differed among the membranes examined. RL and BM demonstrated a significantly higher number of cells at 5 days than at 1.5 hours (p < 0.01). TG had increased numbers of cells at 3 and 5 days after cell seeding. However, there was no statistical difference between the cell numbers at 1.5 hours and 5 days after cell seeding (p > 0.1). EG had a similar number of cell attachments to that at 1.5 hours throughout the experimental period. There was almost no cell proliferation on GT. Cell clusters of ALP positive cells and foci of calcification were seen on all membranes except for GT, where a scant number of cells were seen. Results from this study implied that GTR membrane materials, per se, may influence cell proliferation and differentiation in the process of periodontal tissue regeneration.     

New attachment and bone formation in periodontal defects following treatment of submerged roots with guided tissue regeneration

Abstract The purpose of the present study was to examine the effect on periodontal regeneration of preventing bacterial contamination of the membrane material following the guided tissue regeneration procedure (GTR). Periodontal dehiscence defects were surgically produced in 2 monkeys. In each monkey, 8 of these defects were submerged after resection of the crowns of the teeth and a teflon (Gore-Tex Periodontal Material®) or a polyglactin (Vicryl Mesh®) membrane was adjusted to cover the defect and the exposed root surface. 4 defects on non-crown resected teeth were treated with either a teflon or a polyglactin membrane positioned with the coronal border approximately 2 mm below the margin of the covering tissue flap. Following 6 months of healing, the animals were sacrificed. Histological evaluation of the specimens revealed that roots which were kept completely covered during the healing period demonstrated new connective tissue attachment and bone formation corresponding to 67–100% of the length of the initial defect depth, whereas the amount of new connective tissue attachment and bone on non-submerged roots ranged between 30–59% and 11–31%, respectively. It seems reasonable to anticipate that it is bacterial contamination of the membrane material which jeopardizes the formation of new connective tissue attachment but in particular bone formation following the GTR-procedure.     

Periodontal repair in dogs: effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of alveolar bone and periodontal attachment

OBJECTIVES: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been shown to stimulate alveolar bone and cementum formation in periodontal defects but not a functionally oriented periodontal ligament (PDL). Subcutaneous and intramuscular implants of BMP-12 have been shown to induce tendon formation and ligament-like tissue. The objective of this study was to evaluate rhBMP-12 for periodontal regeneration, in particular PDL formation. METHODS: Six young adult Hound Labrador mongrel dogs were used. Routine supraalveolar periodontal defects were created around the mandibular premolar teeth. Three animals received rhBMP-12(0.04 mg/ml) in an absorbable collagen sponge (ACS) carrier vs. rhBMP-12(0.2 mg/mL)/ACS in contralateral defects. Three animals received rhBMP-12(1.0 mg/ml)/ACS vs. rhBMP-2(0.2 mg/ml)/ACS (total implant volume/defect approximately 1 ml). The animals were euthanized 8 weeks postsurgery and block biopsies were processed for histometric analysis. RESULTS: Bone regeneration appeared increased in sites receiving rhBMP-2/ACS compared to sites receiving rhBMP-12/ACS. Cementum regeneration was similar comparing sites implanted with rhBMP-2/ACS to sites implanted with rhBMP-12/ACS. In contrast, sites receiving rhBMP-12/ACS exhibited a functionally oriented PDL bridging the gap between newly formed bone and cementum whereas this was a rare observation in sites receiving rhBMP-2/ACS. Ankylosis appeared increased in sites receiving rhBMP-2/ACS compared to those receiving rhBMP-12/ACS. CONCLUSIONS: The outcomes of this study suggest that rhBMP-12 may have significant effects on regeneration of the PDL. Additional preclinical evaluation is needed to confirm these initial observations prior to clinical application.     

重度牙周病患牙拔除后即刻自身牙移植2年疗效与体会

目的观察重度牙周病患牙拔除后即刻自身牙移植的临床效果,分析影响临床效果的因素。方法9颗重度牙周病患牙经基础治疗后拔除,去除牙槽窝内的病变组织并以此处牙槽窝为受牙区。微创拔除患者自身第三磨牙(供牙),体外完成根管治疗和充填后植入已制备的牙槽窝内。根周骨腔植入拜阿蒙人工骨,缝合、结扎固定。术后随访观察2年的临床疗效。结果9颗重度牙周病患牙拔除后自身第三磨牙移植2年间除因松动拔除3颗外,其余6颗移植牙稳固、不松动,牙周无红肿,无可探入的牙周袋,可行使正常咀嚼功能。结论重度牙周病患牙拔除后即刻自身牙移植可取得较好的临床效果,去除病变组织、保持移植牙稳定和术后口腔卫生是影响疗效的主要因素。     

Prognostic factors for alveolar regeneration: bone formation at teeth and titanium implants

OBJECTIVES: There is a limited understanding of the effect of defect characteristics on alveolar bone healing. The objectives of this study were to assess the effect of alveolar bone width and space provision on bone regeneration at teeth and titanium implants, and to test the hypothesis that the regenerative potentials at teeth and implants are not significantly different. METHODS: Critical size, 5-6-mm, supra-alveolar, periodontal defects were surgically created in 10 young adult dogs. Similarly, critical size, 5-mm, supra-alveolar, peri-implant defects were created in four dogs. A space-providing expanded polytetrafluoroethylene device was implanted for guided tissue regeneration/guided bone regeneration. The animals were euthanized at 8 weeks postsurgery. Histometric analysis assessed alveolar bone regeneration (height) relative to space provision by the device and the width of the alveolar crest at the base of the defect. Statistical analysis used the linear mixed models. RESULTS: A significant correlation was found between bone width and wound area (r=0.55892, p<0.0001). Generally, bone width and wound area had statistically significant effects on the extent of bone regeneration (p<0.0005 and p<0.0001, respectively). Bone regeneration was linearly correlated with the bone width at periodontal (p<0.001) and implant (p=0.04) sites, and with the wound area at periodontal (p<0.0001) and implant (p=0.03) sites. The relationships of bone regeneration with these two variables were not significantly different between teeth and implants (bone width: p=0.83; wound area: p=0.09). When adjusted for wound area, bone regeneration was significantly greater at periodontal than at implant sites (p=0.047). CONCLUSIONS: The horizontal dimension of the alveolar bone influences space provision. Space provision and horizontal dimension of the alveolar bone appear to be important determinants of bone regeneration at teeth and implants. The extent of alveolar bone formation at implant sites is limited compared with that at periodontal sites.     

Periodontal repair in dogs: guided tissue regeneration enhances bone formation in sites implanted with a coral-derived calcium carbonate biomaterial

BACKGROUND: Previous studies suggest that a bioresorbable calcium carbonate coral implant (CI) supports space provision and bone formation for guided tissue regeneration (GTR). However, it could not be discerned whether observed effects were because of GTR or whether the CI possessed osteoconductive properties enhancing bone formation. The objective of this study was to evaluate bone formation associated with the CI biomaterial in the presence and absence of provisions for GTR. METHODS: Routine, critical size, 6 mm, supra-alveolar periodontal defects were created in 12 young adult Beagle dogs. Five animals received the CI alone (Biocoral 1000). Seven animals received the CI/GTR combination using an expanded polytetrafluoroethylene barrier (GORE-TEX Regenerative Material). The animals were euthanized at 4 weeks postsurgery and tissue blocks of the experimental sites were collected and processed for histometric analysis. RESULTS: Clinical healing was uneventful. The histopathologic and histometric analysis revealed significantly increased bone formation (height and area) in sites receiving the CI/GTR combination compared with CI alone (2.3+/-0.6 versus 1.2+/-0.9 mm; and 3.1+/-0.8 versus 1.2+/-1.1 mm2; p<0.05). The CI biomaterial appeared to be mostly unassociated with new bone formation; the CI particles were observed sequestered in newly formed bone, fibrovascular marrow, and in the supra-alveolar connective tissue. Cementum formation was limited and observed in few sites for both treatment protocols. CONCLUSION: While GTR promoted new bone formation, the CI contributed limited, if any, osteoconductive effects.     

Effect of allogeneic freeze-dried demineralized bone matrix on regeneration of alveolar bone and periodontal attachment in dogs

Abstract. This split-mouth study was designed to evaluate regeneration of alveolar bone and periodontal attachment following implantation of allogeneic. freeze-dried, demineralized bone matrix (DBM). Buccal fenestration defects (6×4 mm) were created on the maxillary canine teeth in 6 beagle dogs. DBM was implanted into one randomly selected defect in each animal. The contralateral defect served as surgical control. Tissue blocks were harvested following a 4-week healing interval and prepared for histometric analysis. DBM was discernible in all implanted defects with limited evidence of bone metabolic activity. The DBM particles appeared invested within a dense connective tissue, often in close contact to the instrumented root. Fenestration defect height averaged 3.8±0.1 and 3.7±0.3mm, total bone regeneration 0.9±0.9 and 0.4±1.2 mm, and total cementum regeneration 2.3±1.5 and 0.6±0.7 mm for DBM and control defects, respectively. Differences with regards to cementum regeneration were statistically significant ( p =0.03). In summary, the results of this study suggest that DBM implants may enhance cementum regeneration in this defect model, and that they have no apparent effect on alveolar bone regeneration. Enhanced cementum regeneration may be possibly be explained by provisions for guided tissue regeneration from the implant suppressing a significant influence of the gingival connective tissue on the healing process. Moreover, a 4-week healing interval appears insufficient for turnover of DBM.