Enamel Matrix Derivative Research Articles

Peptide-Based Bioinspired Approach to Regrowing Multilayered Aprismatic Enamel.

The gradual discovery of functional domains in native enamel matrix proteins has enabled the design of smart bioinspired peptides for tooth enamel mimetics and repair. In this study, we expanded upon the concept of biomineralization to design smaller amelogenin-inspired peptides with conserved functional domains for clinical translation. The synthetic peptides displayed a characteristic nanostructured scaffold reminiscent of ‘nanospheres’ seen in the enamel matrix and effectively controlled apatite nucleation in vitro resulting in the formation of smaller crystallites. Following application of the peptides to sectioned human molar teeth, a robust, oriented, synthetic aprismatic enamel was observed after 7 days of incubation in situ. There was a two-fold increase in the hardness and modulus of the regrown enamel-like apatite layers and an increase in the attachment of the tooth-regrown layer interface compared to control samples. Repeated peptide applications generated multiple enamel-like hydroxyapatite (HAP) layers of limited thickness produced by epitaxial growth in which c-axis oriented nanorods evolved on the surface of native enamel. We conclude that peptide analogues with active domains can effectively regulate the orientation of regenerated HAP layers to influence functional response. Moreover, this enamel biofabrication approach demonstrates the peptide-mediated growth of multiple microscale HAP arrays of organized microarchitecture with potential for enamel repair.

Combining enamel matrix proteins with mechanical stimuli potentiates human periodontal ligament fibroblasts proliferation and periodontium remodeling.

Collagen I (Col-I) and matrix metalloproteinase-1 (MMP-1) have been implicated in the regeneration and remodeling of the periodontium. Studies have shown that enamel matrix proteins (EMPs) and mechanical stimuli can promote the synthesis and degradation, respectively, of Col-I and MMP-1. However, the effects of the combination of EMPs and mechanical stimuli on human periodontal ligament are not known. Our aim was to test the combined effects of EMPs and mechanical stimuli on the proliferation of human periodontal ligament fibroblasts (HPDLFs) and Col-I and MMP-1 mRNA expression. Primary HPDLFs were isolated using an enzyme digestion method. To select the optimum EMP concentration and the optimum magnitude and loading time of mechanical stimuli, HPDLFs were stimulated with gradient concentration of EMPs (0 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL and 200 µg/mL) and mechanical stimuli (0 kPa, 25 kPa, 50 kPa, 100 kPa, and 200 kPa for 0 h, 3 h, 6 h, 12 h, and 24 h), respectively. The cell proliferative response was tested by the MTT assay. The impact of EMPs combined with mechanical stimuli on Col-I and MMP-1 mRNA expression were measured by reverse transcription polymerase chain reaction. 100 µg/mL of EMPs and a 50 kPa mechanical stimulus were chosen as the optimum parameters due to the higher proliferation rates than other doses. The combination of 100 µg/mL of EMPs and a 50 kPa mechanical stimulus significantly stimulated HPDLFs proliferation and increased Col-I and MMP-1 expression levels compared with incubation with two factors alone. We concluded that the combination of EMPs and mechanical stimulus have synergistic effects on cell growth, cell number, collagen turnover, and periodontium remodeling.

MMP20 Overexpression Disrupts Molar Ameloblast Polarity and Migration

Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice (Mmp20+/+Tg+) were assessed by multiscale analyses, combining several approaches from high-resolution micro–computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20+/+Tg+ mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20+/+Tg+ molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20+/+Tg+ mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.

Histological healing after nonsurgical periodontal treatment with enamel matrix derivatives in canine experimental periodontitis.

The histological outcomes after nonsurgical periodontal treatment with enamel matrix derivatives (EMD) remain controversial. The present study evaluated periodontal wound healing after scaling and root planing (SRP) with subgingival application of EMD for treatment of experimental periodontitis. Periodontal breakdown was induced by applying silk ligatures around mandibular third and fourth premolars of six beagle dogs until radiographic bone loss progressed to approximately half of the root length. Probing pocket depth (PPD) and clinical attachment level (CAL) were proximally measured 2weeks after ligature removal (baseline). Mesial and distal surfaces of the experimental teeth were subjected to SRP and randomized using a split-mouth design to subgingival application of EMD (test) or normal saline (control). PPD and CAL were re-evaluated at 11weeks. Animals were sacrificed at 12weeks for histological analyses. No significant differences were observed in PPD and CAL between both groups at baseline and at 11weeks. Histologically, test sites exhibited a greater amount of new cementum than that did the control sites (p<0.01). Moreover, the control sites revealed increased epithelial downgrowth compared with the test sites: (p<0.05). On the other hand, no intergroup differences were detected in terms of bone position, connective tissue attachment, gingival recession, and planed root length. This study suggested that EMD has an increased potential to support formation of new cementum with decreased epithelial downgrowth when used as an adjunct to nonsurgical periodontal treatment.

Effect of EMD on the orthodontically induced root resorption repair process in rats.

While different levels of root resorption may occur in orthodontic treatment, several preventive approaches have been reported. Nevertheless, little is known about the effect of enamel matrix derivative (EMD) on root repair during orthodontic tooth movement. To evaluate the effect of EMD on root resorption repair following the application of orthodontic force. Aforce of 100 g was exerted for 14days on the left maxillary first molars of twenty 10-week-old Sprague-Dawley rats divided into the EMD and control groups (n= 10 per group). In the EMD group, repeatedly injection of Emdogain® was administered after the appliance was removed, while phosphate-buffered saline was administered in the control group. In vivo microcomputed tomography (CT), haematoxylin and eosin (H&E) staining, and immunohistochemistry were then used to evaluate the effect of EMD on the process of root repair. In the EMD group, the observed decrease in root resorption crater volume and increase in both the bone volume fraction and trabecular thickness were significantly greater than those in the control group. H&E staining showed that the periodontal fibres were relatively regular in arrangement and that the surface of the cementum was smooth in the EMD group. Immunohistochemical analysis showed higher bone morphogenetic protein 2 (BMP-2) and bone sialoprotein (BSP) expression levels in the EMD group than in the control group. In addition, the osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) expression levels were similar in both groups. EMD enhanced the repair process following orthodontically induced root resorption in rats.

Using Enamel Matrix Derivative to Improve Treatment Efficacy in Periodontal Furcation Defects.

Furcations are complicated periodontal defects. Untreated furcations lead to loss of the involved teeth and supporting tissues. It has been demonstrated that regenerative biomaterials are beneficial in reconstruction of the bone surrounding furcation-affected teeth. These biomaterials range from bone grafts and nonresorbable/resorbable barrier membranes to biologics that are able to trigger inactive regenerative processes in periodontal tissues. Selection of appropriate material(s) to treat furcations is challenging. The aim of this article is to provide a comparative outlook on different biomaterials applicable in regeneration of furcations with a focus on enamel matrix derivative (EMD). Scientific databases including PubMed/MEDLINE, ScienceDirect, and EMBASE were searched, and 28 articles were found primarily for this specific study. Full texts were studied to identify relevant studies; 17 studies were excluded because of irrelevancy, while 11 main studies were ultimately selected. Other references have been used for general statements. EMD is a protein complex widely used in the regeneration of different periodontal defects. To assess the effects of EMD for treatment of root furcations, clinical studies involving EMD with and without barrier membranes and bone grafts were selected and compared. Briefly, this study reveals that when EMD is combined with open flap debridement (OFD), guided tissue regeneration (GTR), or bone grafting (BG), the amount of class II furcations converted to class I increases significantly. EMD also reduces tissue swelling and patient discomfort after treatment. This study provides evidence to find the best combination of biomaterials to treat furcation defects. The best results are obtained if EMD is combined with β-TCP/HA alloplastic bone grafts.

Amelogenin X impacts age-dependent increase of frequency and number in labial incisor grooves in C57BL/6

Labial grooves in maxillary incisors have been reported in several wild-type rodent species. Previous studies have reported age-dependent labial grooves occur in moderate prevalence in C57BL/6 mice; however, very little is known about the occurrence of such grooves. In the present study, we observed age-dependent groove formation in C57BL/6 mice up to 26 months after birth and found that not only the frequency of the appearance of incisor grooves but also the number of grooves increased in an age-dependent manner. We examined the molecular mechanisms of age-dependent groove formation by performing DNA microarray analysis of the incisors of 12-month-old (12M) and 24-month-old (24M) mice. Amelx, encoding the major enamel matrix protein AMELOGENIN, was identified as a 12M-specific gene. Comparing with wild-type mice, the maxillary incisors of Amelx−/− mutants indicated the increase of the frequency and number of labial grooves. These findings suggested that the Amelx gene impacts the age-dependent appearance of the labial incisor groove in C57BL/6 mice.

A Comparable Study of Combinational Regenerative Therapies Comprising Enamel Matrix Derivative plus Deproteinized Bovine Bone Mineral with or without Collagen Membrane in Periodontitis Patients with Intrabony Defects

Aim: The aim of the present study was to examine the effectiveness of collagen membrane (CM) in regenerative therapy with deproteinized bovine bone mineral (DBBM) and enamel matrix derivative (EMD) for periodontal intrabony defects. Methods: Eighteen periodontal intrabony defects of nine chronic periodontitis patients were evaluated. Two defects per patient with probing pocket depth (PPD) ≥ 6 mm were assigned to two different types of treatments: EMD + DBBM + CM or EMD + DBBM. Clinical parameters including Gingival Index (GI), PPD, clinical attachment level (CAL), gingival recession (GR), bleeding on probing (BOP), tooth mobility (MOB), and the filled bone volume/rate (FBV/FBR), which was measured by cone beam computed tomography, were compared at baseline and 12 months post-treatment. Differences between groups were determined by the chisquare test, McNemar’s test, and Wilcoxon signed-rank test. Results: Clinically, PPD, CAL, and FBR significantly improved in both groups (p < 0.05). The between-group comparison showed that the EMD + DBBM + CM group resulted in slightly greater PPD reduction, CAL gain, and FBR; however, these differences were not statistically significant. Conclusion: Periodontal regenerative therapies comprising EMD and DBBM with and without CM resulted in positive clinical outcomes. The use of CM may result in better outcomes in MOB decrease; however, long-term prognosis must be further studied.

Efficacy of flapless intentional replantation with enamel matrix derivative in the treatment of hopeless teeth associated with endodontic-periodontal lesions: A 2-year prospective case series.

In this prospective case series, the effects of flapless intentional replantation (IR) with root surface conditioning with enamel matrix derivative (EMD) was evaluated for the treatment of periodontally-endodontally involved hopeless teeth. A total of 17 teeth (17 patients, aged 40 to 73) with ≥ 10 mm attachment loss and radiographic bone loss to the apex were included in the study. The affected teeth were atraumatically extracted using a flapless approach, then granulation tissues, calculus, and necrotic cementum on the root surfaces were removed. EMD was applied for 5 minutes to the root surfaces. The teeth were reinserted to the socket without any bone grafts and splinted. After 2 years, clinical healing was uneventful in 16 cases and the radiographs revealed no root resorption or ankylosis. IR with EMD treatment provided statistically significant reduction in probing depth (6.4 ± 2.6 mm), gain in clinical attachment level (5.9 ± 2.5 mm), and gain in radiographic bone level (48.2 ± 26.1%) compared to baseline values (P < .01). Within the limitations of the study, flapless IR with EMD treatment appeared to act as an alternative strategy to extraction of severely compromised teeth with endodontic-periodontal lesions beyond the root apex.

The use of Enamel Matrix Derivative (Emd) for Treatment of Combined Apicomarginal Lesions in Apical Surgery: A Retrospective Analysis.

Objective:The outcome of apical surgery using modern techniques is favourable. However, the presence of a combined apicomarginal defect may negatively affect the postsurgical healing. The objective of this retrospective analysis was to assess the healing of teeth with apicomarginal defects treated with apical surgery and enamel matrix derivative (EMD).Methods:This retrospective study evaluated the application of EMD in apical surgery of 17 teeth with apicomarginal defects. Cases were followed for at least 1 year, and healing was classified based on established clinical and radiographic criteria.Results:The patient sample included nine females and eight males with a mean age of 50±18.2 years. Maxillary incisors (six lateral and four central) were the most frequently treated teeth. The majority of apicomarginal defects was located on the facial aspect of the root (70.6%) and belonged to defect class I (76.5%). Follow-up periods ranged from 1 to 5 years. Healing was successful in 14 teeth (82.4%).Conclusion:The application of EMD resulted in a similar outcome as in previously published clinical studies related to regenerative techniques for the treatment of apicomarginal defects in conjunction with apical surgery.

Comparison of the Influence of a New Synthetic Peptide and Enamel Matrix Derivative in the Early Wound Healing Process of Artificial Periodontal Tissue Defects in Rats

Comparison of the Influence of a New Synthetic Peptide and Enamel Matrix Derivative in the Early Wound Healing Process of Artificial Periodontal Tissue Defects in Rats

Treatment of a vertical bone defect with enamel matrix derivative in the upper molar: A case report

Treatment of a vertical bone defect with enamel matrix derivative in the upper molar: A case report

Nonincised Papillae Surgical Approach (NIPSA) in Periodontal Regeneration: Preliminary Results of a Case Series.

A new surgical approach has been developed to optimize the preservation of the gingival margin and papillae when treating periodontal defects. The flap is raised by one mucosal incision far away from the marginal tissues. This case series reports on the effectiveness of a nonincised surgical approach (NIPSA) in conjunction with a hydroxyapatite-based graft biomaterial and enamel matrix derivative in treating intrabony defects. Ten defects in 10 patients were treated. The follow-up period ranged from 6 to 18 months (mean: 10.8 ± 4.7 months). Probing pocket depth was 9.6 ± 2.3 mm before surgery and 2.3 ± 0.5 mm postsurgery. Clinical attachment level (CAL) decreased from 10.4 ± 2.7 mm to 3.1 ± 0.87 mm postsurgery. The gingival papilla height, keratinized tissue width, and buccal gingival margin remained stable over time. No wound dehiscence was recorded. Mean Early Healing Index was 1.5 ± 0.7. Results show a substantial CAL gain, limited postsurgical shrinkage, minimal morbidity, and early healing.

Clinical usability of aspartate aminotransferase to evaluate the prognosis of periodontal regeneration therapies: prospective, longitudinal study.

To evaluate the degree of periodontal tissue destruction, aspartate aminotransferase (AST) levels in the gingival crevicular fluid (GCF) are utilized as a predictor of periodontal therapy. We have previously shown that the usefulness of AST activities [periodontal tissue monitor (PTM) values] using a PTM-kit to evaluate the effects of initial periodontal therapy and periodontal regeneration therapy by enamel matrix derivative (EMD). This prospective, longitudinal study was conducted using 38 healthy and 80 periodontitis sites with probing depth (PD) of 5-10mm for guided tissue regeneration (GTR) and EMD from 36 patients. GCF samples were used to evaluate PTM values at base line (BL) and after 6months of surgeries (re-evaluation: RE), and periodontal examinations were performed concurrently. PTM values at BL were statistically improved at RE, accompanied by the improvement of periodontal parameters in both groups. PTM values and PD, and the clinical attachment level (CAL) showed high correlations. PD, CAL and bleeding on probing (BOP) were highly correlated with PTM values in both groups, whereas only PD showed a significant correlation with PTM values at RE in the GTR group. Change in the amounts of PD, CAL and BOP between BL and RE in both groups showed no correlation with PTM values. In the negative PTM value sites at BL in EMD group, the mean PD was significantly reduced at RE compared with positive PTM sites at BL. PTM values are able to be utilized as the biochemical predictor of prognosis after periodontal regeneration therapy.

Xenogenous Collagen Matrix and/or Enamel Matrix Derivative for Treatment of Localized Gingival Recessions: A Randomized Clinical Trial. Part I: Clinical Outcomes.

Considering xenogeneic collagen matrix (CM) and enamel matrix derivative (EMD) characteristics, it is suggested that their combination could promote superior clinical outcomes in root coverage procedures. Thus, the aim of this parallel, double-masked, dual-center, randomized clinical trial is to evaluate clinical outcomes after treatment of localized gingival recession (GR) by a coronally advanced flap (CAF) combined with CM and/or EMD. Sixty-eight patients presenting one Miller Class I or II GRs were randomly assigned to receive either CAF (n = 17); CAF + CM (n = 17); CAF + EMD (n = 17), or CAF + CM + EMD (n = 17). Recession height, probing depth, clinical attachment level, and keratinized tissue width and thickness were measured at baseline and 90 days and 6 months after surgery. The obtained root coverage was 68.04% ± 24.11% for CAF; 87.20% ± 15.01% for CAF + CM; 88.77% ± 20.66% for CAF + EMD; and 91.59% ± 11.08% for CAF + CM + EMD after 6 months. Groups that received biomaterials showed greater values (P <0.05). Complete root coverage (CRC) for CAF + EMD was 70.59%, significantly superior to CAF alone (23.53%); CAF + CM (52.94%), and CAF + CM + EMD (51.47%) (P <0.05). Keratinized tissue thickness gain was significant only in CM-treated groups (P <0.05). The three approaches are superior to CAF alone for root coverage. EMD provides highest levels of CRC; however, the addition of CM increases gingival thickness. The combination approach does not seem justified.

Xenogenous Collagen Matrix and/or Enamel Matrix Derivative for Treatment of Localized Gingival Recessions: A Randomized Clinical Trial. Part II: Patient-Reported Outcomes.

Gingival recession (GR) might be associated with patient discomfort due to cervical dentin hypersensitivity (CDH) and esthetic dissatisfaction. The aim is to evaluate the effect of root coverage procedure with a xenogenous collagen matrix (CM) and/or enamel matrix derivative (EMD) in combination with a coronally advanced flap (CAF) on CDH, esthetics, and oral health-related quality of life (OHRQoL) of patients with GR. Sixty-eight participants with single Miller Class I/II GRs were treated with CAF (n = 17), CAF + CM (n = 17), CAF + EMD (n = 17), and CAF + CM + EMD (n = 17). CDH was assessed by evaporative stimuli using a visual analog scale (VAS) and a Schiff scale. Esthetics outcome was assessed with VAS and the Questionnaire of Oral Esthetic Satisfaction. Oral Health Impact Profile-14 (OHIP-14) questionnaire was used to assess OHRQoL. All parameters were evaluated at baseline and after 6 months. Intragroup analysis showed statistically significant reduction in CDH and esthetic dissatisfaction with no intergroup significant differences (P >0.05). The impact of oral health on QoL after 6 months was significant for CAF + CM, CAF + EMD, and CAF + CM + EMD (P <0.05). Total OHIP-14 score and psychologic discomfort, psychologic disability, social disability, and handicap dimensions showed negative correlation with esthetics. OHIP-14 physical pain dimension had positive correlation with CDH (P <0.05). OHIP-14 showed no correlation with percentage of root coverage, keratinized tissue width, or keratinized tissue thickness (P >0.05). Root coverage procedures improve patient OHRQoL by impacting on a wide range of dimensions, perceived after reduction of CDH and esthetic dissatisfaction of patients with GRs treated with CAF + CM, CAF + EMD, and CAF + CM + EMD.

Enamel matrix proteins regulate hypoxia-induced cellular biobehavior and osteogenic differentiation in human periodontal ligament cells

Hypoxia is a crucial microenvironment for inflamed periodontal tissue and periodontal wound healing. Enamel matrix proteins (EMPs) potentially can promote the formation of new periodontium. The effects of EMPs on periodontal ligament cells under hypoxia, however, remain unclear. We investigated the effects of EMPs on cellular biobehavior and osteogenic differentiation of human periodontal ligament cells (hPDLCs) under hypoxia. Under cobalt chloride (CoCl2)-induced hypoxia, cellular biobehavior of hPDLCs, including proliferation, attachment, spreading, and migration with or without EMPs, was evaluated by 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), cell counting, spreading area measurement and wound scratch assay. The osteogenic activity of hPDLCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining (ARS). The expressions of osteogenic genes including runt related transcription factor 2 (Runx2), ALP, osteocalcin (OCN) and collagen type I (Col-I) were detected using real time quantitative PCR, western blot and immunocytochemistry assays. The biobehavior and osteogenic differentiation of hPDLCs were inhibited significantly under hypoxia. EMPs have no effect on cell proliferation under mimicked hypoxia. EMPs partly reversed the inhibitory effects of hypoxia, however, for other cellular biobehavior including attachment, spreading and migration, and markedly up-regulated osteogenic differentiation activities including ALP, mineralization ability and the expressions of osteogenic genes such as Runx2, ALP, osteocalcin, and collagen type I in hPDLCs under hypoxia. EMPs attenuate the hypoxic injury to cellular biobehavior and osteogenic differentiation in hPDLCs under hypoxia.

A novel volumetric analysis using cad/cam scanners in gingival recession treatment

Objective: In this study, randomized clinical trial (RCT), the aim was to compare clinical periodontal data of subepithelial connective tissue graft (SCTG) group and SCGT+enamel matrix derivatives (EMD) group in terms of gained gingiva volume (GGV) and gained gingiva surface area (GGSA) by scanners of computer-aided design/computer-aided manufacturing (CAD/CAM) devices. Material and Methods: In this study, a total of 35 Miller class I-II gingival recession defects were involved (n=17 in SCTG group, n=18 in SCTG+EMD group). In addition to, periodontal clinical parameters, and three dimensional (3D) images were recorded by using CAD/CAM system before the treatment, at the 1st month and the 6th months after the treatment. The images were superimposed by using softwares and GGV, and GGSA were calculated. Results: There was no significant difference in terms of GGV and GGSA values at the 1st month and 6thmonths between SCTG+EMD and SCTG groups. Similarly, there was no significant difference in periodontal clinical parameters between groups. Conclusion: Images obtained from CAD/CAM system can be easily used in calculating GGV and GGSA data after the gingival recession treatment and in determining of the success of administered treatment. It is expected that the evaluation of the success of many therapeutic approaches for periodontal treatment will be carried out by using CAD/CAM system in the future.

Integrative Temporo-Spatial, Mineralogic, Spectroscopic, and Proteomic Analysis of Postnatal Enamel Development in Teeth with Limited Growth.

Tooth amelogenesis is a complex process beginning with enamel organ cell differentiation and enamel matrix secretion, transitioning through changes in ameloblast polarity, cytoskeletal, and matrix organization, that affects crucial biomineralization events such as mineral nucleation, enamel crystal growth, and enamel prism organization. Here we have harvested the enamel organ including the pliable enamel matrix of postnatal first mandibular mouse molars during the first 8 days of tooth enamel development to conduct a step-wise cross-sectional analysis of the changes in the mineral and protein phase. Mineral phase diffraction pattern analysis using single-crystal, powder sample X-ray diffraction analysis indicated conversion of calcium phosphate precursors to partially fluoride substituted hydroxyapatite from postnatal day 4 (4 dpn) onwards. Attenuated total reflectance spectra (ATR) revealed a substantial elevation in phosphate and carbonate incorporation as well as structural reconfiguration between postnatal days 6 and 8. Nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) demonstrated highest protein counts for ECM/cell surface proteins, stress/heat shock proteins, and alkaline phosphatase on postnatal day 2, high counts for ameloblast cytoskeletal proteins such as tubulin β5, tropomyosin, β-actin, and vimentin on postnatal day 4, and elevated levels of cofilin-1, calmodulin, and peptidyl-prolyl cis-trans isomerase on day 6. Western blot analysis of hydrophobic enamel proteins illustrated continuously increasing amelogenin levels from 1 dpn until 8 dpn, while enamelin peaked on days 1 and 2 dpn, and ameloblastin on days 1–5 dpn. In summary, these data document the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis from a systems biological perspective, including (i) relatively high levels of matrix protein expression during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8. Together, this study provides a baseline for a comprehensive understanding of the mineralogic and proteomic events that contribute to the complexity of mammalian tooth enamel development.

Effect of the combined use of enamel matrix derivative and atelocollagen sponge scaffold on osteoblastic differentiation of mouse induced pluripotent stem cells in vitro.

Induced pluripotent stem cells (iPSCs) are a candidate cell source in periodontal regenerative therapy. Enamel matrix derivative (EMD) has been shown to regenerate periodontal tissues, and atelocollagen sponge (ACS) is considered a suitable scaffold or carrier for growth factors. This study aimed to investigate the effect of combined use of EMD and an ACS scaffold on cell behaviors and differentiation of mouse iPSCs (miPSCs) in vitro. Following embryonic body formation from miPSCs, dissociated cells (miPS-EB-derived cells) were seeded onto ACS with or without EMD, and cultured in osteoblast differentiation medium. Scanning electron microscopy and histological analyses were used to assess cell morphology and infiltration within the ACS. Cell viability (metabolism) was determined using an MTS assay, and expression of mRNA of osteoblastic differentiation markers was assessed by quantitative RT -PCR. Alkaline phosphatase (ALP) staining intensity and activity were evaluated. Mineralization was assessed by von Kossa staining, and calcium content was quantitated using the methylxylenol blue method. By 24hours after seeding, miPS-EB-derived cells in both the EMD and control groups had attached to and infiltrated the ACS scaffold. Scanning electron microscopy images revealed that by day 14, many cytoplasmic protrusions and extracellular deposits, suggestive of calcified matrix, were present in the EMD group. There was a time-dependent increase in cell viability up to day 3, but no difference between groups was observed at any time point. The levels expressed of ALP and osterix genes were significantly higher in the EMD group than in the control group. Expression of runt-related transcription factor 2 was increased in the EMD group compared with the control group on day 7. EMD upregulated the expression of bone sialoprotein and osteopontin on day 14, whereas expression of osteocalcin was lower at all time points. The staining intensity and activity of ALP were higher in the EMD group than in the control group. Mineralization levels and calcium contents were significantly higher in the EMD group throughout the observation period. These data suggest that combining ACS with EMD increases levels of osteoblastic differentiation and mineralization in miPS-EB-derived cells, compared with ACS used alone.