Germ Formation Research Articles

Histological and immunohistochemical characteristic of the gingival stroma in the portion of the third molars in children of various age.

Aim: To evaluate the state of the gingival stromal elements in the portion of the third molars requiring extraction of these teeth due to orthodontic indications considering the stage of tooth germ formation. Materials and Methods: The surgery to extract third molars due to orthodontic indications was performed on 95 children aged 11 to 18 years. The three groups of observation were isolated according to clinical-radiological signs: І (n=30) - children aged 11-13 years; ІІ (n=35) - children aged 13-16 years, and ІІІ (n=30) - children aged 16-18 years. During surgery, the samples of gums were taken from the adjacent areas for examination. The samples were fixed, dehydrated, paraffinized for further histological processing. Immunohistochemical methods were used according to the protocols supplied by a producer. In particular, by means of immunohistochemical method, Ki-67, CD-34 antigens and vimentin with primary antibodies against them were determined. The primary antibodies were visualized by the polymeric visualization system with diaminobenzidine giving a brown color to the places of location of the antigens examined. The data obtained were statistically processed. Results: The results of the study showed that specific gravity of the vascular bed in the gingival papillary layer of children was the most variable. It ranges from (12,7±0,09) % at the stage of "D" root formation to (54,8±0,17) % at the "H" stage. Lower concentrations of CD-34 antigens and vimentin are found in the endotheliocytes of children aged 13-16 and 16-18 years, compared to the children aged 11-13 years (p<0,05). No changes were found in the specific volume of the blood vessels, CD-34 antigens and vimentin in the reticular gingival layer of children from the groups of observation. Conclusions: Therefore, the conducted histological and immunohistochemical study of the connective gingival tissues in the portion of the third molars in children enables to draw a conclusion that in the process of formation of the root of this tooth a number of changes occur in the gingival stroma. They include an increase of the blood flow volume in the papillary gingival layer on the background of a decreased concentration of CD-34 genes and vimentin, a longer stage of development of the third molar root. The specific volume of the islets of neoangiogenesis of the papillary gingival layer is the largest in children aged 13-16 years.

Keratin-mediated hair growth and its underlying biological mechanism

Here we show that intradermal injection of keratin promotes hair growth in mice, which results from extracellular interaction of keratin with hair forming cells. Extracellular application of keratin induces condensation of dermal papilla cells and the generation of a P-cadherin-expressing cell population (hair germ) from outer root sheath cells via keratin-mediated microenvironmental changes. Exogenous keratin-mediated hair growth is reflected by the finding that keratin exposure from transforming growth factor beta 2 (TGFβ2)-induced apoptotic outer root sheath cells appears to be critical for dermal papilla cell condensation and P-cadherin-expressing hair germ formation. Immunodepletion or downregulation of keratin released from or expressed in TGFβ2-induced apoptotic outer root sheath cells negatively influences dermal papilla cell condensation and hair germ formation. Our pilot study provides an evidence on initiating hair regeneration and insight into the biological function of keratin exposed from apoptotic epithelial cells in tissue regeneration and development.

A randomized trial of topical mitomycin-C in the immediate post-operative period for the short and long-term prevention of granulation tissue in mastoid cavities

BackgroundGranulation tissue is a primary source of otorrhea in the ear, particularly after surgery, and may develop complications in patients. Hence, discovering an efficient treatment is crucial for this disorder. PurposeThis study aimed to evaluate the mitomycin drops efficacy for germination prevention during the recovery process after mastoidectomy. Our purpose was to introduce novel treatment options in some conditions in which we could not reconstitute the cavity to prevent the possible germination. Materials and methodsThis clinical trial was performed on 52 patients undergoing mastoidectomy. Patients were randomly divided into two groups (26 patients in the mitomycin group and 26 patients in the placebo group). After drug administration, the granulation tissue and complications were recorded. All statistical analysis was performed using SPSS version 21. ResultsThe results of our study showed that mitomycin administration in patients undergoing mastoidectomy significantly reduced the formation of the granulation tissue in the first and third months after surgery (P < 0.05), which is associated with increased patient satisfaction (P < 0.05). ConclusionThe germ formation after mastoidectomy is so common. Since mitomycin administration decreased the incidence of germ formation, it can be proposed as a suggestive treatment in all patients to increase surgical quality and decrease complications include granulation formation.

An in situ hybridization study of syndecan family during the late stages of developing mouse molar tooth germ.

Expression of syndecan-1, 2, 3, and 4 mRNAs during the late stages of tooth germ formation was investigated by in situ hybridization, using [35S]-UTP-labeled cRNA probes. Syndecan-1 mRNA was mainly expressed in the stellate reticulum and stratum intermedium as well as at the cervical region of dental papilla/dental follicle during E18.5-P3.0. Expression in the dental epithelium was enhanced during the postnatal periods, which was supported by real-time RT-PCR analysis. These spatiotemporal expression patterns may suggest specific roles of syndecan-1 in tooth formation such as tooth eruption or root formation. Syndecan-3 mRNA expression became evident in odontoblasts at E18.5, but compared to collagen type I mRNA, which was strongly expressed at this stage, syndecan-3 expression in odontoblast was restricted in mature odontoblasts beneath the cusps during the postnatal periods. This result was also supported by real-time RT-PCR analysis, and indicated that syndecan-3 may be involved in the progress of dentinogenesis rather than in the initiation of it. Syndecan-4 mRNA roughly showed comparable expression patterns to those of syndecan-3. Syndecan-2 mRNA did not show significant expression during the experimental period, but real-time RT-PCR analysis suggested that syndecan-2 expression might be enhanced with hard tissue formation.

Developmental role of hyaluronic acid and its application in salivary gland tissue engineering

Dry mouth, or xerostomia, caused by salivary gland dysfunction significantly impacts oral/systemic health and quality of life. Although in vitro-generated artificial salivary glands have been considered as the fundamental solution, its structural complexity is difficult to reproduce using current biomaterials. Therefore, understanding and recapitulating the roles of biomacromolecules in salivary gland organogenesis is needed to solve these problems. Hyaluronic acid (HA) is a macromolecule abundant during salivary gland organogenesis, but its role remains unknown. Here, we verify the effects of HA on salivary gland organogenesis and artificial organ germ formation in solubilized and substrate-immobilized forms. In embryonic submandibular glands (eSMG), we found dense HA layers encapsulating proliferative c-Kit+ progenitor cells that were expressing CD44, an HA receptor. The blockage of HA synthesis, or degradation of HA, impaired eSMG growth by ablating the c-Kit+ progenitor cell population. We also found that high-molecular-weight (HMW) HA has a significant role in eSMG growth. Based on these findings, we discovered that HA is also crucial for in vitro formation of salivary gland organ germs, one of the most promising candidates for salivary gland tissue regeneration. We significantly enhanced salivary gland organ germ formation by supplementing HMW HA in solution; this effect was further increased when the HMW HA was immobilized on the substrate by polydopamine/HA co-immobilization. Our study suggests that the current use of HA in salivary gland tissue engineering can be further optimized.

Functional ectodermal organ regeneration as the next generation of organ replacement therapy.

In this decade, substantial progress in the fields of developmental biology and stem cell biology has ushered in a new era for three-dimensional organ regenerative therapy. The emergence of novel three-dimensional cell manipulation technologies enables the effective mimicking of embryonic organ germ formation using the fate-determined organ-inductive potential of epithelial and mesenchymal stem cells. This advance shows great potential for the regeneration of functional organs with substitution of complete original function in situ. Organoids generated from multipotent stem cells or tissue stem cells via establishment of an organ-forming field can only partially recover original organ function owing to the size limitation; they are considered ‘mini-organs’. Nevertheless, they hold great promise to realize regenerative medicine. In particular, regeneration of a functional salivary gland and an integumentary organ system by orthotopic and heterotopic implantation of organoids clearly points to the future direction of organ regeneration research. In this review, we describe multiple strategies and recent progress in regenerating functional three-dimensional organs, focusing on ectodermal organs, and discuss their potential and future directions to achieve organ replacement therapy as a next-generation regenerative medicine.

MOVEMENT SPECIFICS OF A GERM WITH COMPLEX SHAPE ON THE PELLETIZING DISK IN IRON-ORE PELLETS PRODUCTION

The movement specifics of a germ with complex shape on the pelletizing disk in production of iron-ore pellets are considered. As a result of the analysis of the forces acting on the cubic germ during its roll on the charge scull (CS) in the conditions of plastic deformation, a mathematical model was build that allows the optimal parameters of the pelletizer to be determined for the production of pellets based on the technology of a forced germ formation. Two particular cases of germ stability on the pelletizer CS and the conditions for the over-coming of the stability associated with the formation of a roll are considered. The germ stability, or conversely, its roll is determined by the horizontal position of its mass center relative to the support edge located across the probable direction of the roll. If the line of the acting force (gravity force) moves behind the support edge and the reference plane, a force moment will be generated which will lead the germ out of the stability state and form a germ roll. The first special case of germ stability on the inclined plane is the absence of a roll on the CS with a minimum value of the centrifugal force corresponding to the location of the germ in the central areas of the pelletizing zone or to operation of a working element with a low rotation speed ( n 3 rpm) and location of the germ in peripheral areas of the working zone of the pelletizer. For these cases the conditions for germs roll on the CS in the pelletizer working zone and correcting measures in the conditions of plastic deformation during the corners and edges crushing are formulated. The features of the germ movement in the working zone of the pelletizing disk are shown. The conditions for correcting the regime of forced germ formation are provided, and the measures are indicated for the purposeful impact on the crushing factor of a germ. It was established that for the organization of the regime of a germ roll with complex shape the angle of disk inclination to the horizon should be set depending on the frequency of its rotation and the crushing factor of germs.

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine

Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

CALCIFYING CYSTIC ODONTOGENIC TUMOR ASSOCIATED WITH ODONTOMA: A CASE REPORT

The calcifying cystic odontogenic tumor is a rare developmental lesion that arises from the odontogenic epithelium. An 8-year-old female patient, featuring swelling involving mandible body region. Radiographic examination revealed presence of radiolucent image, circumscribed and involving teeth without root resorption. The histopathologic analysis showed large cystic cavities lined by thin epithelium that was thickened in other areas, with cells featuring increased eosinophilia with ghost cells characteristics and points with dystrophic calcifications. It was observed cords and nests of columnar epithelial cells that are arranged parallel on the periphery. It is noted still that dental germ formation consists of stellate cells layers to enamel organ, odontoblasts layer with dental matrix production and areas with fusiform cells which suggest dental papilla. Based on clinical, radiographic and histologic, the diagnosis of Calcifying Cystic Odontogenic Tumor associated to Odontoma was confirmed. The patient is under observation and after 1 year of follow-up, there is no evidence of recurrence.

Different expression patterns of Lin28 and Lin28b in mouse molar development

ObjectiveThe RNA-binding proteins Lin28 and Lin28b are expressed in many developing tissues and are involved in the biosynthesis of the microRNA let-7 family and embryogenesis processes. However, their roles in mammalian tooth development remain ill-defined. DesignThe spatiotemporal expressions of Lin28 and Lin28b during mouse molar odontogenesis from day E11.5 to P21 were examined through immunohistochemistry and western blot analysis. ResultsBoth Lin28 and Lin28b were initially expressed in dental epithelium, but the expression patterns varied thereafter. Lin28 was expressed in tooth germ from early embryonic stages and was consistently expressed in the ameloblasts and odontoblasts throughout all stages of tooth development. However, positive staining of Lin28b gradually faded out with tooth germ development, before finally disappearing in tooth organ cells after birth. ConclusionsThese results indicate that Lin28 was spatiotemporally expressed in tooth germ throughout tooth development progression and may play an active role in ameloblast and odontoblast differentiation, as well as matrix secretion and the mineralization of enamel and dentin. Its paralogue Lin28b may have a distinct function in tooth germ formation.

Analysis of kinetics pattern in the formation and separation of a drop of fluid in the form of a capsule

Capsulation, as a technological principle, can provoke the activation of innovations in the food industry and cause the development and implementation of new scientifically substantiated technologies for processing the raw materials, creating new product shapes, convenient in the consumption, increasing production volumes and efficiency of food products, development and application of modern fundamentally new technologies, technological processes, techniques and equipment that collectively are able to significantly affect the state and development of food technologies. Taking into account the laws of gravity and the use of methods of systems analysis, we devised a model of capsule-formation of fluids, different in origin, by the method of extrusion. Kinetics of the formation of a capsular structure is defined, as well as the patterns for obtaining the spherical shapes of different diameter. The obtained regularities are the basis of scientific and technological principles of obtaining the capsulated oil and fat products with the thermo- and acid stable properties. It is demonstrated theoretically that the main factor that limits the process of formation and separation of a drop is the stage of germ formation and a drop in particular. Duration of the germ and drop formation is much longer (by about 20 times) than the time of a bridge breakaway. The presence in capsulated fluids of a shell significantly affects both the dimensions of a bridge and a drop and the time of the processes of forming a drop and its separation. In this case, an increase in the relative coefficient of surface tension by 3 times increases the radius of a drop by 1.6 times, and the overall time of the formation separation of a drop by 2.5 times. Resulting equations might be applied in the experimental verification of the proposed model for the formation and separation of a drop of fluid.

MiRNAs Are Required for Postinduction Stage Sweat Gland Development

miRNAs Are Required for Postinduction Stage Sweat Gland Development

Deletion of the epidermis derived laminin γ1 chain leads to defects in the regulation of late hair morphogenesis

Laminins are the most abundant non-collagenous basement membrane (BM) components, composed of an α, β and γ chain. The laminin γ1 chain, encoded by LAMC1, is the most abundant γ chain. The main laminin isoforms in the dermo-epidermal junction (DEJ) are laminin-332, laminin-511 and laminin-211, the latter being restricted to the lower part of hair follicles (HFs). Complete deletion of LAMC1 results in lethality around embryonic day 5.5. To study the function of laminin γ1 containing isoforms in skin development and maturation after birth, we generated mice lacking LAMC1 expression in basal keratinocytes (LAMC1EKO) using the keratin 14 (K14) Cre/loxP system. This deletion resulted in loss of keratinocyte derived laminin-511 and in deposition of fibroblast derived laminin-211 throughout the whole DEJ. The DEJ in areas between hemidesmosomes was thickened, whereas hemidesmosome morphology was normal. Most strikingly, LAMC1EKO mice showed delayed HF morphogenesis accompanied by reduced proliferation of hair matrix cells and impaired differentiation of hair shafts (HS). However, this deletion did not interfere with early HF development, since placode numbers and embryonic hair germ formation were not affected. Microarray analysis of skin revealed down regulation of mainly different hair keratins. This is due to reduced expression of transcription factors such as HoxC13, FoxN1, FoxQ1 and Msx2, known to regulate expression of hair keratins. While the role of laminin-511 in signaling during early hair germ formation and elongation phase has been described, we here demonstrate that epidermal laminin-511 is also a key regulator for later hair development and HS differentiation.

Characterization of novel MSX1 mutations identified in Japanese patients with nonsyndromic tooth agenesis.

Since MSX1 and PAX9 are linked to the pathogenesis of nonsyndromic tooth agenesis, we performed detailed mutational analysis of these two genes sampled from Japanese patients. We identified two novel MSX1 variants with an amino acid substitution within the homeodomain; Thr174Ile (T174I) from a sporadic hypodontia case and Leu205Arg (L205R) from a familial oligodontia case. Both the Thr174 and Leu205 residues in the MSX1 homeodomain are highly conserved among different species. To define possible roles of mutations at these amino acids in the pathogenesis of nonsyndromic tooth agenesis, we performed several functional analyses. It has been demonstrated that MSX1 plays a pivotal role in hard tissue development as a suppressor for mesenchymal cell differentiation. To evaluate the suppression activity of the variants in mesenchymal cells, we used the myoD-promoter, which is one of convenient reporter assay system for MSX1. Although the gene products of these MSX1 variants are stable and capable of normal nuclear localization, they do not suppress myoD-promoter activity in differentiated C2C12 cells. To clarify the molecular mechanisms underlying our results, we performed further analyses including electrophoretic mobility shift assays, and co-immunoprecipitation assays to survey the molecular interactions between the mutant MSX1 proteins and the oligonucleotide DNA with MSX1 consensus binding motif or EZH2 methyltransferase. Since EZH2 is reported to interact with MSX1 and regulate MSX1 mediated gene suppression, we hypothesized that the T174I and L205R substitutions would impair this interaction. We conclude from the results of our experiments that the DNA binding ability of MSX1 is abolished by these two amino acid substitutions. This illustrates a causative role of the T174I and L205R MSX1 homeodomain mutations in tooth agenesis, and suggests that they may influence cell proliferation and differentiation resulting in lesser tooth germ formation in vivo.

Itm2a expression in the developing mouse first lower molar, and the subcellular localization of Itm2a in mouse dental epithelial cells.

Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6) cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.

Many Paths to Alopecia via Compromised Regeneration of Hair Follicle Stem Cells

Alopecia can be caused by defective formation, defective regeneration, or increased destruction of hair follicles. Much work has elucidated the roles of diffusible morphogens in modulating hair follicle stem cell activities. Recent studies have revealed novel molecular events within the nucleus, which are required for the activation and progression of hair stem cells. These studies will provide new clues and targets for designing therapeutic strategies for hair loss.

Porocarcinoma in situ showing follicular differentiation: A case report

Abstract Poroid neoplasm is a skin appendage tumor that has both benign and malignant counterparts. It has traditionally been regarded as of eccrine origin and has four types: intraepidermal poroma (hidroacanthoma simplex), poroma, dermal duct tumor, and poroid hidradenoma. Here we describe the case of a 64-year-old woman who had a verrucous, erythematous to brownish tumor on her left buttock for many years. Histopathology revealed an intra-epidermal poroid tumor with both benign and malignant parts. The benign part had intra-epidermal nests of poroid cells, which were smaller, monomorphic and sharply marginated from adjacent keratinocytes. The malignant part showed similar cell types, but had a higher nuclear/cytoplasmic ratio, pleomorphism, and prominent mitoses. Ductal structures were noted in neoplastic cells and an epithelial membrane antigen stain was strongly positive. Interestingly, peripheral palisading and primitive follicular germ formation were also observed in the neoplasm, which suggests follicular differentiation. We made a final diagnosis of porocarcinoma in situ with follicular differentiation, which may support the folliculosebaceous–apocrine unit theory, but a tumor with such a combination has not been described before.

Size dependence of volume and surface nucleation rates for homogeneous freezing of supercooled water droplets

Abstract. The relative roles of volume and surface nucleation were investigated for the homogeneous freezing of pure water droplets. Experiments were carried out in a cryogenic laminar aerosol flow tube using supercooled water aerosols with maximum volume densities at radii between 1 and 3 μm. Temperature- and size-dependent values of volume- and surface-based homogeneous nucleation rates between 234.8 and 236.2 K were derived using a microphysical model and aerosol phase compositions and size distributions determined from infrared extinction measurements in the flow tube. The results show that the contribution from nucleation at the droplet surface increases with decreasing droplet radius and dominates over nucleation in the bulk droplet volume for droplets with radii smaller than approximately 5 μm. This is interpreted in terms of a lowered free energy of ice germ formation in the surface-based process. The implications of surface nucleation for the parameterization of homogeneous ice nucleation in numerical models are considered.

Parameterizing ice nucleation rates using contact angle and activation energy derived from laboratory data

Abstract. The rate of ice nucleation in clouds is not easily determined and large discrepancies exist between model predictions and actual ice crystal concentration measured in clouds. In an effort to improve the parameterization of ice nucleating in cloud models, we investigate the rate of heterogeneous ice nucleation under specific ambient conditions by knowing the sizes as well as two thermodynamic parameters of the ice nuclei – contact angle and activation energy. Laboratory data of freezing and deposition nucleation modes were analyzed to derive inversely the two thermodynamic parameters for a variety of ice nuclei, including mineral dusts, bacteria, pollens, and soot particles. The analysis considered the Zeldovich factor for the adjustment of ice germ formation, as well as the solute and curvature effects on surface tension; the latter effects have strong influence on the contact angle. Contact angle turns out to be a more important factor than the activation energy in discriminating the nucleation capabilities of various ice nuclei species. By extracting these thermodynamic parameters, laboratory results can be converted into a formulation that follows classical nucleation theory, which then has the flexibility of incorporating factors such as the solute effect and curvature effect that were not considered in the experiments. Due to various uncertainties, contact angle and activation energy derived in this study should be regarded as "apparent" thermodynamics parameters.

Immunocytochemical localization of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 of the human deciduous molar tooth germ development in the human fetus

Vascular endothelial growth factor (VEGF) is a key regulator of blood vessel endothelial development. We used immunohistochemical methods to demonstrate the localization of VEGF and its receptors, showing the specific expression pattern of VEGF and VEGF receptor in the human deciduous tooth from the cap to late bell stages in the human fetus. Immunoreactivity to VEGF and its receptor VEGF receptor-2 (VEGFR-2) was intensely positive in the inner enamel epithelium at the cap stage and ranged from negative to moderately positive in the bell stage. At the late bell stage, VEGF immunoreactivity was mainly positive but weak for VEGFR-2. The intensity of VEGF and VEGFR-2 in odontoblasts increases from cap stage to late bell stage. We postulate that the dissimilar expression of VEGF in inner enamel epithelium, ameloblast and odontoblast during each stage of human tooth development may affect tooth germ formation.