Fogeredetű őssejtek izolálása és jellemzése = Isolation and characterisation of postnatal stem cells of dental origin

Kutatásainkhoz a K61543 kutatási pályázatmellé elnyertük a IN67250 nemzetközi kiegészítő támogatást is, így beszámolónk ezek összefoglaló zárójelentése. Vizsgálataink célja emberi fogbélből és parodontális ligamentumból származó őssejtek izolálása és jellemzése, in vitro modell-rendszerek és eljárások kidolgozása a fogeredetű, és így potenciálisan a fogak és a parodontális szövetek részleges vagy teljes regenerációjára felhasználható őssejtek azonosítására, izolálására és fejlődési, differenciálódási képességeik meghatározására, illetve ectodermális sejtekkel való kölcsönhatásaik jellemzésére. Munkánk során emberi fogak pulpájából (dental pulp stem cell=DPSC) és a parodontális ligamentumból (periodontal ligament stem cells=PDLSC) illetve emberi nyálmirigyekből (PTHSG) preparáltunk pluripotens őssejteket tartalmazó sejtkultúrákat. Ezen kultúrákat molekuláris és sejtbiológiai módszerekkel jellemeztük, a sejteket különböző irányú differenciálódásra / transdifferenciálódásra bírtuk. Az osteogén differenciálódás mellett ki kell emelnünk mind a DPSC, mind a PDLSC sejtek képességét a neuronális differenciálódásra az általunk kidolgozott három lépésből álló protokoll mellett. Fentieken túl egy új állatkísérletes tesztrendszert dolgoztunk ki az osseointegráció, illetve a parodontális ligamentum és a fogbél regenerációjának tanulmányozására. Mindezek megalapozzák további ilyen irányú kutatásainkat, az emberi fogeredetű sejtek in vivo regenerációs alkalmazásának kidolgozására. | The originally supported K61543 research grant received additional support by IN67250, which is a supplementary international extension of the same project. Therefore, this is a joint report of the twin grants. The purpose of our study was to isolate and characterize human stem cells from dental pulp and periodontal ligament, to develop in vitro model systems and processes, for identification of stem cells, which have the potential for full or partial regeneration dental and periodontal tissues. On the course of our work we prepared cultures containing pluripotent postnatal stem cells from the dental pulp (dental pulp stem cell=DPSC), from the periodontal ligament (periodontal ligament stem cells=PDLSC), and from the salivary glands (PTHSG). We characterized these cultures and developed methods for their differentiation / transdifferentiation in vitro. Besides the osteogenic differentiation we must highlight the potency of both DPSC and PDLSC cells for neuronal differentiation when our newly developed three step protocol is used. Moreover we also developed and in vivo animal test model for studying osseointegration, periodontal and pulp regeneration. These results will give the foundation of our future work towards the application of human stem cells of dental origin in biological regeneration of damaged tissues

Downregulation of FGF Signaling by Spry4 Overexpression Leads to Shape Impairment, Enamel Irregularities, and Delayed Signaling Center Formation in the Mouse Molar.

FGF signaling plays a critical role in tooth development, and mutations in modulators of this pathway produce a number of striking phenotypes. However, many aspects of the role of the FGF pathway in regulating the morphological features and the mineral quality of the dentition remain unknown. Here, we used transgenic mice overexpressing the FGF negative feedback regulator Sprouty4 under the epithelial keratin 14 promoter (K14-Spry4) to achieve downregulation of signaling in the epithelium. This led to highly penetrant defects affecting both cusp morphology and the enamel layer. We characterized the phenotype of erupted molars, identified a developmental delay in K14-Spry4 transgenic embryos, and linked this with changes in the tooth developmental sequence. These data further delineate the role of FGF signaling in the development of the dentition and implicate the pathway in the regulation of tooth mineralization. © 2019 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

The composition of the dental pellicle: an updated literature review

BackgroundThe dental pellicle is a thin layer of up to several hundred nm in thickness, covering the tooth surface. It is known to protect the teeth from acid attacks through its selective permeability and it is involved in the remineralization process of the teeth. It functions also as binding site and source of nutrients for bacteria and conditioning biofilm (foundation) for dental plaque formation.MethodsFor this updated literature review, the PubMed database was searched for the dental pellicle and its composition.ResultsThe dental pellicle has been analyzed in the past years with various state-of-the art analytic techniques such as high-resolution microscopic techniques (e.g., scanning electron microscopy, atomic force microscopy), spectrophotometry, mass spectrometry, affinity chromatography, enzyme-linked immunosorbent assays (ELISA), and blotting-techniques (e.g., western blot). It consists of several different amino acids, proteins, and proteolytic protein fragments. Some studies also investigated other compounds of the pellicle, mainly fatty acids, and carbohydrates.ConclusionsThe dental pellicle is composed mainly of different proteins, but also fatty acids, and carbohydrates. Analysis with state-of-the-art analytical techniques have uncovered mainly acidic proline-rich proteins, amylase, cystatin, immunoglobulins, lysozyme, and mucins as main proteins of the dental pellicle. The pellicle has protective properties for the teeth. Further research is necessary to gain more knowledge about the role of the pellicle in the tooth remineralization process

Enamel Research: Mechanisms and Characterization

The rodent incisor is a good model system to study the molecular and cellular events that are involved in enamel biomineralization. Incisors in rodents continuously erupt during their lifespan, thus allowing the study of all stages of enamel synthesis, deposition, mineralization and maturation in the same tissue section. This model system has provided invaluable insight into the specifics of enamel formation as a basis to understand human pathologies such as amelogenesis imperfect. Furthermore, the rodent incisor allows exploration and understanding of some of the most fundamental mechanisms that govern biomineralization. Enamel is the most mineralized, hardest tissue in the body. It is formed within a unique organic matrix that, unlike other hard tissues such as bone and dentin, does not contain collagen. The formation of enamel can be divided into two main stages: the secretory and maturation stage. During the secretory stage, a highly ordered arrangement of hydroxyapatite crystals is formed under the influence of structural matrix proteins such as amelogenin, ameloblastin and enamelin. During the maturation stage, the organic matrix is removed and hydroxyapatite crystals expand to ultimately yield a functional hard structure consisting of over 96% mineral. Research efforts over the past decades have mainly focused on the secretory stage, providing novel insights into the concept of biomineralization. However, the events that occur during the maturation stage have not been yet explored in detail, likely because the physiological roles of the enamel-forming ameloblasts are more diverse and complex at this stage. Mature ameloblasts are involved in the regulation of calcium transport in large amounts, phosphate and protein fragments in and out of the maturing enamel and provide regulatory mechanisms for the control of the pH. In recent years, increased efforts have been dedicated towards defining the molecular events during enamel maturation. The development of an ever-increasing number of transgenic animal models has clearly demonstrated the essential roles of matrix and non-matrix proteins during enamel formation. Multiple traditional and modern analytical techniques are applied for the characterization of enamel in these animals. The need for this Research Topic therefore stems from new information that has been generated on molecular events during the enamel maturation stage and the development and application of highly advanced analytical techniques to characterize dental enamel. The benefits and limitations of these techniques need to be reviewed and their application standardized for valid comparative studies

Flaxseed enhances the beneficial effect of low-dose estrogen therapy at reducing bone turnover and preserving bone microarchitecture in ovariectomized rats.

Our previous research showed greatest protection to vertebral bone mineral density and strength in ovariectomized (OVX) rats when lignan- and alpha-linolenic acid-rich flaxseed (FS) is combined with low-dose estrogen therapy (LD) compared to either treatment alone. This study determined the effects of combined FS+LD on serum and tissue markers of bone turnover and microarchitecture to explain our previous findings. 3-month-old OVX rats were randomized to negative control (NEG), FS, LD or FS+LD for 2 or 12 weeks, meaningful time points for determining effects on markers of bone metabolism and bone structure, respectively. Ground FS was added to the AIN-93M diet (100g/kg diet) and LD (0.42μg 17β-estradiol/kg body weight/day) was delivered by subcutaneous implant. Sham rats were included as positive control. Bone formation (e.g. osteocalcin), bone resorption (e.g. tartrate-resistant acid phosphatase-5β (TRAP-5β)), as well as osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were analyzed from the 2-week study by commercial assays (serum) and/or histology (vertebra). Vertebral bone microarchitecture was measured from the 12-week study using microcomputed tomography. In serum, FS+LD and LD induced lower TRAP-5β and osteocalcin, and higher OPG and OPG/RANKL ratio versus NEG and FS (p<0.05). In vertebrae, FS+LD induced higher OPG and lower osteocalcin versus NEG (p<0.01) and did not differ from LD and FS. FS+LD improved bone microarchitecture versus NEG, FS and LD (p<0.05). In conclusion, FS+LD protects bone tissue due to a reduction in bone turnover. However, elucidating the distinctive action of FS+LD on bone turnover compared to LD requires further investigation

Evaluation of Amelotin Expression in Benign Odontogenic Tumors

Objective: Amelotin (AMTN) is highly and selectively expressed by odontogenic epithelium-derived ameloblasts throughout the maturation stage of enamel formation. The protein is secreted and concentrated at the basal lamina interface between ameloblasts and the mineralized enamel matrix. Odontogenic tumors (OT) are characterized by morphological resemblance to the developing tooth germ. OT vary from slowly expanding, encapsulated tumors to locally aggressive and destructive lesions. The purpose of this study was to determine the expression profile of AMTN in benign odontogenic tumors and to correlate it with specific features of the lesions. Methods: Immunohistochemical staining for AMTN was performed on human ameloblastoma, ameloblastic fibroma (AF), ameloblastic fibro-odontoma (AFO), odontoma, adenomatoid odontogenic tumor (AOT) and calcifying cystic odontogenic tumor (CCOT). Results: Generally, ameloblastoma and AF did not stain for AMTN. A strong signal was detected in ameloblast-like layers of AFO and odontoma. Epithelial cells in AOT did not stain for AMTN, while calcifying areas of extracellular eosinophilic matrix were intensely stained. Interestingly, ghost cells present in odontomas and CCOT revealed variable staining, again in association with calcification foci. Conclusions: Amelotin expression was consistently detected in tumors presenting differentiated ameloblasts and obvious matrix deposition. Additionally, the presence of the protein in the eosinophilic matrix and small mineralized foci of AOT and calcification areas of ghost cells may suggest a role for AMTN in the control of mineralization events. [J Interdiscipl Histopathol 2013; 1(5.000): 236-245

A Highly Conserved Motif within the Amelotin Protein Controls the Surface Growth of Brushite

Amelotin (AMTN) protein exerts a direct role on enamel biomineralization likely due to its binding affinity with calcium phosphates (Ca-Ps). However, the kinetics and molecular mechanisms of the AMTN–Ca-P interaction remain largely unknown. Here we used in situ atomic force microscopy (AFM) to directly image the surface growth of brushite (dicalcium phosphate dihydrate, DCPD, CaHPO₄·2H₂O) in the presence of recombinant human AMTN. Measured step movement velocities of the DCPD (010) face show that AMTN protein promotes crystal face growth only within a limited concentration range, whereas inhibition occurs outside of this range. A peptide derived from a highly conserved and potentially phosphorylated motif (SSEEL) within the AMTN protein inhibits crystal growth similar to that of the AMTN protein at low concentration. By the use of single-molecule force spectroscopy (SMFS), we directly measure the binding of the full-length AMTN and SSEEL to the DCPD (010) face. Similar rupture forces reveal that this active SSEEL subdomain may contribute to a specific interaction with the DCPD (010) face, despite significant differences in binding energies of the full-length AMTN and SSEEL peptides to the DCPD surfaces. The findings reveal the kinetic and energetic basis for modulation of the Ca–P crystal face growth by AMTN and provide first evidence for a functional subdomain that is critical in controlling enamel biomineralization

Natural graft tissues and synthetic biomaterials for periodontal and alveolar bone reconstructive applications: a review

Abstract Periodontal disease is categorized by the destruction of periodontal tissues. Over the years, there have been several clinical techniques and material options that been investigated for periodontal defect repair/regeneration. The development of improved biomaterials for periodontal tissue engineering has significantly improved the available treatment options and their clinical results. Bone replacement graft materials, barrier membranes, various growth factors and combination of these have been used. The available bone tissue replacement materials commonly used include autografts, allografts, xenografts and alloplasts. These graft materials mostly function as osteogenic, osteoinductive and/or osteoconductive scaffolds. Polymers (natural and synthetic) are more widely used as a barrier material in guided tissue regeneration (GTR) and guided bone regeneration (GBR) applications. They work on the principle of epithelial cell exclusion to allow periodontal ligament and alveolar bone cells to repopulate the defect before the normally faster epithelial cells. However, in an attempt to overcome complications related to the epithelial down-growth and/or collapse of the non-rigid barrier membrane and to maintain space, clinicians commonly use a combination of membranes with hard tissue grafts. This article aims to review various available natural tissues and biomaterial based bone replacement graft and membrane options used in periodontal regeneration applications

Targeted Overexpression of Amelotin Disrupts the Microstructure of Dental Enamel

We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes ’ process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation o