Dental stem cells: The role of biomaterials and scaffolds in developing novel therapeutic strategies

Dental stem cells(DSCs) are self-renewable cells that can be obtained easily from dental tissues, and are a desirable source of autologous stem cells. The use of DSCs for stem cell transplantation therapeutic approaches is attractive due to their simple isolation, high plasticity, immunomodulatory properties, and multipotential abilities. Using appropriate scaffolds loaded with favorable biomolecules, such as growth factors, and cytokines, can improve the proliferation, differentiation, migration, and functional capacity of DSCs and can optimize the cellular morphology to build tissue constructs for specific purposes. An enormous variety of scaffolds have been used for tissue engineering with DSCs. Of these, the scaffolds that particularly mimic tissue-specific micromilieu and loaded with biomolecules favorably regulate angiogenesis, cell-matrix interactions, degradation of extracellular matrix, organized matrix formation, and the mineralization abilities of DSCs in both in vitro and in vivo conditions. DSCs represent a promising cell source for tissue engineering, especially for tooth, bone, and neural tissue restoration. The purpose of the present review is to summarize the current developments in the major scaffolding approaches as crucial guidelines for tissue engineering using DSCs and compare their effects in tissue and organ regeneration.     

Intraocular neovascularization.

An important character of the eye is transparency, so intraocular neovascularization, which is fragile and likely to result in hemorrhage, would cause a functional disorder of the eye and contribute to loss of vision associated with such diseases as retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration. Recently interest in the mechanisms of intraocular neovascularization has increased, and the mechanisms have been gradually elucidated using several in vitro and in vivo angiogenesis models. Blood vessels in the eye are composed of, and surrounded by, various types of cells that produce multiple factors. Neovascularization is regulated by complex interactions among these angiogenic factors, angiostatic factors, and adhesion molecules, and some of these angiogenesis-related molecules have also been suggested as new targets for novel therapeutic agents of intraocular neo-vascularization. This review focuses on in vivo representative angiogenesis models of the corneal pocket model and the model of oxygen-induced retinopathy, and discusses the role of some angiogenesis-related factors and adhesion molecules in intraocular neovascularization.     

T helper cell-dependent, microbial superantigen-induced murine B cell activation: polyclonal and antigen-specific antibody responses.

Microbial superantigens (SA), bound to human B cell surface MHC class II molecules, have been shown to promote direct, "cognate" interaction with SA-reactive autologous Th cells, resulting in polyclonal Ig production. To investigate the potential for microbial SA to support Th cell-dependent, Ag-specific antibody responses, we have extended our studies to the murine system. BALB/c Th cell lines (TCL), specific for either the Mycoplasma arthritis-derived SA or the Staphylococcus aureus-derived toxic shock syndrome toxin-1) were generated. These TCL cells are SA-specific, functionally noncross-reactive, and utilize distinct TCR V beta gene families. Coculture of SA-reactive TCL cells and syngeneic B cells bearing the relevant SA results in B cell proliferation and polyclonal IgM and IgG production. In contrast, Ag-specific (SRBC-specific) antibody-forming cells are only generated in cultures that also contain SRBC. Thus, microbial SA-mediated Th-B cell interactions induce both polyclonal B cell activation and provide selective help for the proliferation and/or differentiation of B cells that have encountered specific Ag. In additional studies, we determined that the in vivo administration of toxic shock syndrome toxin-1 to young, athymic (nude) BALB/c mice results in SA binding to splenic B cells, rendering these B cells effective stimulators of and targets for SA-reactive helper TCL cells. Taken together, these results demonstrate that microbial SA mediate productive Th-B cell interactions analogous to those that occur during allospecific Th-B cell interactions in vitro and GVHD in vivo. These findings are consistent with the hypothesis that microbial SA represent environmental factors that may trigger autoimmune disease in the genetically susceptible host.     

A photo-cross-linking approach to monitor protein dynamics in living cells

BackgroundProteins, which comprise one of the major classes of biomolecules that constitute a cell, interact with other cellular factors during both their biogenesis and functional states. Studying not only static but also transient interactions of proteins is important to understand their physiological roles and regulation mechanisms. However, only a limited number of methods are available to analyze the dynamic behaviors of proteins at the molecular level in a living cell. The site-directed in vivo photo-cross-linking approach is an elegant technique to capture protein interactions with high spatial resolution in a living cell.Scope of reviewHere, we review the in vivo photo-cross-linking approach including its recent applications and the potential problems to be considered. We also introduce a new in vivo photo-cross-linking-based technique (PiXie) to study protein dynamics with high spatiotemporal resolution.Major conclusionsIn vivo photo-cross-linking enables us to capture weak/transient protein interactions with high spatial resolution, and allows for identification of interacting factors. Moreover, the PiXie approach can be used to monitor rapid folding/assembly processes of proteins in living cells.General significanceIn vivo photo-cross-linking is a simple method that has been used to analyze the dynamic interactions of many cellular proteins. Originally developed in Escherichia coli, this system has been extended to studies in various organisms, making it a fundamental technique for investigating dynamic protein interactions in many cellular processes. This article is part of a Special issue entitled “Novel major techniques for visualizing ‘live’ protein molecules” edited by Dr. Daisuke Kohda.     

Poisson-Boltzmann continuum-solvation models: applications to pH-dependent properties of biomolecules

All molecules can be viewed as either discrete or continuous assemblies of electric charges, and electrostatics plays a major role in intermolecular and intramolecular interactions. Moreover, charge distribution within molecules may fluctuate due to the presence of ionizable groups capable of exchanging protons with the environment, leading to pH-dependence of phenomena involving such molecules. Electrostatic aspects of complex shapes and environments of biological molecules, in vitro and in vivo, are relatively well amenable to treatment by Poisson-Boltzmann models, which are attractive in that they possess a clear physical meaning, and can be readily solved by several mathematically sound methods. Here we describe applications of these models to obtain valuable insights into some biologically important pH-dependent properties of biomolecules, such as stability, binding of ligands (including potential drugs), enzymatic activity, conformational transitions, membrane transport and viral entry.     

Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach

Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes.     

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.     

Fitness Factors in Vibrios: a Mini-review

Vibrios are Gram-negative curved bacilli that occur naturally in marine, estuarine, and freshwater systems. Some species include human and animal pathogens, and some vibrios are necessary for natural systems, including the carbon cycle and osmoregulation. Countless in vivo and in vitro studies have examined the interactions between vibrios and their environment, including molecules, cells, whole animals, and abiotic substrates. Many studies have characterized virulence factors, attachment factors, regulatory factors, and antimicrobial resistance factors, and most of these factors impact the organism's fitness regardless of its external environment. This review aims to identify common attributes among factors that increase fitness in various environments, regardless of whether the environment is an oyster, a rabbit, a flask of immortalized mammalian cells, or a planktonic chitin particle. This review aims to summarize findings published thus far to encapsulate some of the basic similarities among the many vibrio fitness factors and how they frame our understanding of vibrio ecology. Factors representing these similarities include hemolysins, capsular polysaccharides, flagella, proteases, attachment factors, type III secretion systems, chitin binding proteins, iron acquisition systems, and colonization factors.     

Prion-Prion Interactions

The term prion has been used to describe self-replicating protein conformations that can convert other protein molecules of the same primary structure into its prion conformation. Several different proteins have now been found to exist as prions in Saccharomyces cerevisiae. Surprisingly, these heterologous prion proteins have a strong influence on each others’ appearance and propagation, which may result from structural similarity between the prions. Both positive and negative effects of a prion on the de novo appearance of a heterologous prion have been observed in genetic studies. Other examples of reported interactions include mutual or unilateral inhibition and destabilization when two prions are present together in a single cell. In vitro work showing that one purified prion stimulates the conversion of a purified heterologous protein into a prion form, suggests that facilitation of de novo prion formation by heterologous prions in vivo is a result of a direct interaction between the prion proteins (a cross-seeding mechanism) and does not require other cellular components. However, other cellular structures, e.g., the cytoskeleton, may provide a scaffold for these interactions in vivo and chaperones can further facilitate or inhibit this process. Some negative prion-prion interactions may also occur via a direct interaction between the prion proteins. Another explanation is a competition between the prions for cellular factors involved in prion propagation or differential effects of chaperones stimulated by one prion on the heterologous prions.     

Communication between white cells and the abnormalities of this in leukemia.

The production of white blood cells is mediated by cellular communication. Proliferation and differentiation of granulocytic and monocytic progenitor and immature cells have been studied in detail and are regulated by stimulatory and inhibitory molecules produced and released from the progeny of the progenitor cells. The resultant interactions between cells and cell-derived molecules suggest operable positive and negative feed-back mechanisms during normal hematopoiesis, and what one sees in the end is the net result of these interactions. The complexities of cellular regulation are partially unravelled by physical separation of the different populations and biochemical analysis of the regulatory molecules. Subpopulations of granulocyte-monocyte progenitors (CFU-c) exist and evidence suggests that they vary in responsiveness to different molecules. Stimulatory molecules are themselves chemically and physically heterogenous and, until recently, believed to have similar biological actions, but this concept must be re-evaluated. Different molecules may activate different subsets of progenitor cells, and there is now a role for substances which enhance the stimulatory interactions. Many studies on normal and leukemic cell responsiveness to stimulation must be re-examined in light of this recent information. Several inhibitory substances operate during normal hematopoiesis. Mature granulocytes, progeny of CFU-c, appear to elaborate at least two inhibitory activities. One activity influences immature, recognizable granulocytes and the other indirectly reduces progenitor cell proliferation by decreasing the production and release of molecules which stimulate CFU-c. In addition, mononuclear phagocytes produce and release E-type prostaglandins in direct response to elevated levels of the stimulatory molecules and E-type prostaglandins counteract increased stimulatory levels by decreasing the sensitivity of CFU-c to stimulators. Much of our present level of sophistication derives from in vitro experimentation and it is apparent that we are only beginning to understand these inter-relationships and their relevance to the in vivo situation. However, these in vitro studies have shed light on the interactions occurring during leukemia. Leukemic cells retain the capacity to respond to normal regulators and must therefore be considered dependent rather than autonomous neoplasms. Abnormalities do exist in leukemic cell interactions: the progenitor cells themselves may be defective and leukemic cells may respond to molecules which normal cells do not. The degree of sensitivity to stimulators and inhibitors will have to be carefully investigated to determine if and what differences may exist between normal and leukemic cells. Normal mature granulocyte derived inhibitory activity is quantitatively deficient in leukemic cells but another inhibitory activity which appears to be specifically present in cells from patients with leukemia and some cases of myelodysplasia is present...     

Growth factors and beta cell replication

Recent studies have demonstrated that human islet allograft transplantation can be a successful therapeutic option in the treatment of patients with Type I diabetes. However, this impressive recent advance is accompanied by a very important constraint. There is a critical paucity of pancreatic islets or pancreatic beta cells for islet transplantation to become a large-scale therapeutic option in patients with diabetes. This has prompted many laboratories around the world to invigorate their efforts in finding ways for increasing the availability of beta cells or beta cell surrogates that potentially could be transplanted into patients with diabetes. The number of studies analyzing the mechanisms that govern beta cell proliferation and growth in physiological and pathological conditions has increased exponentially during the last decade. These studies exploring the role of growth factors, intracellular signaling molecules and cell cycle regulators constitute the substrate for future strategies aimed at expanding human beta cells in vitro and/or in vivo after transplantation. In this review, we describe the current knowledge on the effects of several beta cell growth factors that have been shown to increase beta cell proliferation and expand beta cell mass in vitro and/or in vivo and that they could be potentially deployed in an effort to increase the number of patients transplanted with islets. Furthermore, we also analyze in this review recent studies deciphering the relevance of these specific islet growth factors as physiological and pathophysiological regulators of beta cell proliferation and islet growth.     

Biocompatible controlled release polymers for delivery of polypeptides and growth factors

The development of biocompatible, controlled release systems for macromolecules has provided the opportunity for researchers and clinicians to target and deliver, on site, biologically active factors. This advance has also facilitated the purification and characterization of a number of important biomolecules. These systems include controlled release delivery systems which release proteins through porous polymer matrices, degradable polymeric delivery systems, and modulated polymer release systems. These areas of research will be reviewed with regards to their design, release kinetics, and biocompatibilities. The utilization of these systems to release such biologically important polypeptides as growth factors (e.g., fibroblast growth factor, epidermal growth factor, transforming growth factor-B) as well as a number of important inhibitory factors (e.g., nitrosoureas, angiogenesis inhibitors) in both in vivo and in vitro studies will be discussed.     

Invited review: mechanochemical signal transduction in the fetal lung.

Growth and maturation of fetal lungs are regulated by both humoral and physical factors. Mechanical stretch stimulates fetal lung cell proliferation and affects fetal lung maturation by influencing the production of extracellular matrix molecules and the expression of specific genes of fetal lung cells. These effects are mediated through special signal transduction pathways in fetal lung cells. Various in vivo and in vitro model systems have been developed to investigate the mechanotransduction process. The diversity and discrepancy of these studies have raised many questions. We will briefly summarize mechanical force-induced signals in fetal lung cell proliferation and differentiation and then discuss several important issues related to these studies.