Exosomes: A New Hope for Angiogenesis-Mediated Bone Regeneration.

Bone is a metabolically dynamic structure that is generally remodeled throughout the lifetime of an individual but often causes problems with increasing age. A key player for bone development and homeostasis, but also under pathological conditions, is the bone vasculature. This complex system of arteries, veins, and capillaries forms distinct structures where each subset of endothelial cells has important functions. Starting with the basic process of angiogenesis and bone-specific blood vessel formation, coupled with initial bone formation, the importance of different vascular structures is highlighted with respect to how these structures are maintained or changed during homeostasis, aging, and pathological conditions. After exemplifying the current knowledge on bone vasculature, this review will move on to exosomes, a novel hotspot of scientific research. Exosomes will be introduced starting from their discovery via current isolation procedures and state-of-the-art characterization to their role in bone vascular development, homeostasis, and bone regeneration and repair while summarizing the underlying signal transduction pathways. With respect to their role in these processes, especially mesenchymal stem cell-derived extracellular vesicles are of interest, which leads to a discussion on patented applications and an update on ongoing clinical trials. Taken together, this review provides an overview of bone vasculature and bone regeneration, with a major focus on how exosomes influence this intricate system, as they might be useful for therapeutic purposes in the near future.

Therapeutic Treatments for Osteoporosis-Which Combination of Pills Is the Best among the Bad?

Osteoporosis is a chronical, systemic skeletal disorder characterized by an increase in bone resorption, which leads to reduced bone density. The reduction in bone mineral density and therefore low bone mass results in an increased risk of fractures. Osteoporosis is caused by an imbalance in the normally strictly regulated bone homeostasis. This imbalance is caused by overactive bone-resorbing osteoclasts, while bone-synthesizing osteoblasts do not compensate for this. In this review, the mechanism is presented, underlined by in vitro and animal models to investigate this imbalance as well as the current status of clinical trials. Furthermore, new therapeutic strategies for osteoporosis are presented, such as anabolic treatments and catabolic treatments and treatments using biomaterials and biomolecules. Another focus is on new combination therapies with multiple drugs which are currently considered more beneficial for the treatment of osteoporosis than monotherapies. Taken together, this review starts with an overview and ends with the newest approaches for osteoporosis therapies and a future perspective not presented so far.

Sinking Our Teeth in Getting Dental Stem Cells to Clinics for Bone Regeneration.

Dental stem cells have been isolated from the medical waste of various dental tissues. They have been characterized by numerous markers, which are evaluated herein and differentiated into multiple cell types. They can also be used to generate cell lines and iPSCs for long-term in vitro research. Methods for utilizing these stem cells including cellular systems such as organoids or cell sheets, cell-free systems such as exosomes, and scaffold-based approaches with and without drug release concepts are reported in this review and presented with new pictures for clarification. These in vitro applications can be deployed in disease modeling and subsequent pharmaceutical research and also pave the way for tissue regeneration. The main focus herein is on the potential of dental stem cells for hard tissue regeneration, especially bone, by evaluating their potential for osteogenesis and angiogenesis, and the regulation of these two processes by growth factors and environmental stimulators. Current in vitro and in vivo publications show numerous benefits of using dental stem cells for research purposes and hard tissue regeneration. However, only a few clinical trials currently exist. The goal of this review is to pinpoint this imbalance and encourage scientists to pick up this research and proceed one step further to translation.

P2 Receptors Influence hMSCs Differentiation towards Endothelial Cell and Smooth Muscle Cell Lineages.

BACKGROUND: Human mesenchymal stem cells (hMSCs) have shown their multipotential including differentiating towards endothelial and smooth muscle cell lineages, which triggers a new interest for using hMSCs as a putative source for cardiovascular regenerative medicine. Our recent publication has shown for the first time that purinergic 2 receptors are key players during hMSC differentiation towards adipocytes and osteoblasts. Purinergic 2 receptors play an important role in cardiovascular function when they bind to extracellular nucleotides. In this study, the possible functional role of purinergic 2 receptors during MSC endothelial and smooth muscle differentiation was investigated. METHODS AND RESULTS: Human MSCs were isolated from liposuction materials. Then, endothelial and smooth muscle-like cells were differentiated and characterized by specific markers via Reverse Transcriptase-PCR (RT-PCR), Western blot and immunochemical stainings. Interestingly, some purinergic 2 receptor subtypes were found to be differently regulated during these specific lineage commitments: P2Y4 and P2Y14 were involved in the early stage commitment while P2Y1 was the key player in controlling MSC differentiation towards either endothelial or smooth muscle cells. The administration of natural and artificial purinergic 2 receptor agonists and antagonists had a direct influence on these differentiations. Moreover, a feedback loop via exogenous extracellular nucleotides on these particular differentiations was shown by apyrase digest. CONCLUSIONS: Purinergic 2 receptors play a crucial role during the differentiation towards endothelial and smooth muscle cell lineages. Some highly selective and potent artificial purinergic 2 ligands can control hMSC differentiation, which might improve the use of adult stem cells in cardiovascular tissue engineering in the future.

Tooth Formation: Are the Hardest Tissues of Human Body Hard to Regenerate?

With increasing life expectancy, demands for dental tissue and whole-tooth regeneration are becoming more significant. Despite great progress in medicine, including regenerative therapies, the complex structure of dental tissues introduces several challenges to the field of regenerative dentistry. Interdisciplinary efforts from cellular biologists, material scientists, and clinical odontologists are being made to establish strategies and find the solutions for dental tissue regeneration and/or whole-tooth regeneration. In recent years, many significant discoveries were done regarding signaling pathways and factors shaping calcified tissue genesis, including those of tooth. Novel biocompatible scaffolds and polymer-based drug release systems are under development and may soon result in clinically applicable biomaterials with the potential to modulate signaling cascades involved in dental tissue genesis and regeneration. Approaches for whole-tooth regeneration utilizing adult stem cells, induced pluripotent stem cells, or tooth germ cells transplantation are emerging as promising alternatives to overcome existing in vitro tissue generation hurdles. In this interdisciplinary review, most recent advances in cellular signaling guiding dental tissue genesis, novel functionalized scaffolds and drug release material, various odontogenic cell sources, and methods for tooth regeneration are discussed thus providing a multi-faceted, up-to-date, and illustrative overview on the tooth regeneration matter, alongside hints for future directions in the challenging field of regenerative dentistry.

Non-Cytotoxic Agarose/Hydroxyapatite Composite Scaffolds for Drug Release.

Healing of large bone defects requires implants or scaffolds that provide structural guidance for cell growth, differentiation, and vascularization. In the present work, an agarose-hydroxyapatite composite scaffold was developed that acts not only as a 3D matrix, but also as a release system. Hydroxyapatite (HA) was incorporated into the agarose gels in situ in various ratios by a simple procedure consisting of precipitation, cooling, washing, and drying. The resulting gels were characterized regarding composition, porosity, mechanical properties, and biocompatibility. A pure phase of carbonated HA was identified in the scaffolds, which had pore sizes of up to several hundred micrometers. Mechanical testing revealed elastic moduli of up to 2.8 MPa for lyophilized composites. MTT testing on Lw35human mesenchymal stem cells (hMSCs) and osteosarcoma MG-63 cells proved the biocompatibility of the scaffolds. Furthermore, scaffolds were loaded with model drug compounds for guided hMSC differentiation. Different release kinetic models were evaluated for adenosine 5'-triphosphate (ATP) and suramin, and data showed a sustained release behavior over four days.

Small Molecules Enhance Scaffold-Based Bone Grafts via Purinergic Receptor Signaling in Stem Cells.

The need for bone grafts is high, due to age-related diseases, such as tumor resections, but also accidents, risky sports, and military conflicts. The gold standard for bone grafting is the use of autografts from the iliac crest, but the limited amount of accessible material demands new sources of bone replacement. The use of mesenchymal stem cells or their descendant cells, namely osteoblast, the bone-building cells and endothelial cells for angiogenesis, combined with artificial scaffolds, is a new approach. Mesenchymal stem cells (MSCs) can be obtained from the patient themselves, or from donors, as they barely cause an immune response in the recipient. However, MSCs never fully differentiate in vitro which might lead to unwanted effects in vivo. Interestingly, purinergic receptors can positively influence the differentiation of both osteoblasts and endothelial cells, using specific artificial ligands. An overview is given on purinergic receptor signaling in the most-needed cell types involved in bone metabolism-namely osteoblasts, osteoclasts, and endothelial cells. Furthermore, different types of scaffolds and their production methods will be elucidated. Finally, recent patents on scaffold materials, as wells as purinergic receptor-influencing molecules which might impact bone grafting, are discussed.

Lignin-Derived Biomaterials for Drug Release and Tissue Engineering.

Renewable resources are gaining increasing interest as a source for environmentally benign biomaterials, such as drug encapsulation/release compounds, and scaffolds for tissue engineering in regenerative medicine. Being the second largest naturally abundant polymer, the interest in lignin valorization for biomedical utilization is rapidly growing. Depending on its resource and isolation procedure, lignin shows specific antioxidant and antimicrobial activity. Today, efforts in research and industry are directed toward lignin utilization as a renewable macromolecular building block for the preparation of polymeric drug encapsulation and scaffold materials. Within the last five years, remarkable progress has been made in isolation, functionalization and modification of lignin and lignin-derived compounds. However, the literature so far mainly focuses lignin-derived fuels, lubricants and resins. The purpose of this review is to summarize the current state of the art and to highlight the most important results in the field of lignin-based materials for potential use in biomedicine (reported in 2014⁻2018). Special focus is placed on lignin-derived nanomaterials for drug encapsulation and release as well as lignin hybrid materials used as scaffolds for guided bone regeneration in stem cell-based therapies.

The conditioned medium from osteo-differentiating human mesenchymal stem cells affects the viability of triple negative MDA-MB231 breast cancer cells.

This study aimed to investigate the effect of conditioned media (CM) from osteo-differentiating and adipo-differentiating human mesenchymal stem cells (MSCs) isolated from lipoaspirates of healthy female donors on the viability of triple-negative breast cancer cells MDA-MB231. The CM of undifferentiated and differentiating MSCs were collected after 7, 14, 21 and 28 days of culture. The effects of MSC CM on cell proliferation were assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24 h. The effects of osteo-differentiating cell CM on apoptotic promotion, cell cycle impairment, mitochondrial transmembrane potential dissipation, production of reactive oxygen species and autophagosome accumulation were analysed by flow cytometry and Western blot. MTT assay showed that only CM collected from osteo-induced cells at day 28 (d28O-CM) reduced tumour cell viability. Treatment with d28O-CM restrained cell cycle progression through G2 phase, elicited a caspase-8-driven apoptotic effect already after 5 h of culture, and down-regulated autophagosome accumulation and beclin-1 expression. The finding that factor(s) secreted by osteo-differentiating MSCs shows properties of an apoptotic inducer and autophagy inhibitor on triple-negative breast cancer cells may have an important applicative potential that deserves further investigation.

Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors.

Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 µM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe's concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 µM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes' lipophilicity, and the obtained

Type V collagen counteracts osteo-differentiation of human mesenchymal stem cells.

In search of novel gene signatures for osteo-differentiation of mesenchymal stem cells (MSCs), we submitted cDNA preparations from undifferentiated and differentiating MSCs to differential display- and semiquantitative-PCR and found down-regulation of COL5A1 in osteo-induced cultures at days 21 and 28, when the mineralized matrix accumulates. We also cultured osteo-differentiating MSCs onto type V collagen substrates and found a decrease in the accumulation of extracellular calcium compared to those grown in uncoated flasks. To our knowledge, this is first evidence that type V collagen might represent a stromal component that impairs osteogenesis.

Essential roles of Raf/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway, YY1, and Ca2+ influx in growth arrest of human vascular smooth muscle cells by bilirubin.

The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition.

Mechanisms underlying the osteo- and adipo-differentiation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are considered a promising cell source for regenerative medicine, because they have the potential to differentiate into a variety of lineages among which the mesoderm-derived lineages such adipo- or osteogenesis are investigated best. Human MSCs can be harvested in reasonable to large amounts from several parts of the patient's body and due to this possible autologous origin, allorecognition can be avoided. In addition, even in allogenic origin-derived donor cells, hMSCs generate a local immunosuppressive microenvironment, causing only a weak immune reaction. There is an increasing need for bone replacement in patients from all ages, due to a variety of reasons such as a new recreational behavior in young adults or age-related diseases. Adipogenic differentiation is another interesting lineage, because fat tissue is considered to be a major factor triggering atherosclerosis that ultimately leads to cardiovascular diseases, the main cause of death in industrialized countries. However, understanding the differentiation process in detail is obligatory to achieve a tight control of the process for future clinical applications to avoid undesired side effects. In this review, the current findings for adipo- and osteo-differentiation are summarized together with a brief statement on first clinical trials.

Effects of Silicon Compounds on Biomineralization, Osteogenesis, and Hard Tissue Formation.

Bioinspired stem cell-based hard tissue engineering includes numerous aspects: The synthesis and fabrication of appropriate scaffold materials, their analytical characterization, and guided osteogenesis using the sustained release of osteoinducing and/or osteoconducting drugs for mesenchymal stem cell differentiation, growth, and proliferation. Here, the effect of silicon- and silicate-containing materials on osteogenesis at the molecular level has been a particular focus within the last decade. This review summarizes recently published scientific results, including material developments and analysis, with a special focus on silicon hybrid bone composites. First, the sources, bioavailability, and functions of silicon on various tissues are discussed. The second focus is on the effects of calcium-silicate biomineralization and corresponding analytical methods in investigating osteogenesis and bone formation. Finally, recent developments in the manufacturing of Si-containing scaffolds are discussed, including in vitro and in vivo studies, as well as recently filed patents that focus on the influence of silicon on hard tissue formation.

Polysaccharide-Based Systems for Targeted Stem Cell Differentiation and Bone Regeneration.

Bone tissue engineering is an ever-changing, rapidly evolving, and highly interdisciplinary field of study, where scientists try to mimic natural bone structure as closely as possible in order to facilitate bone healing. New insights from cell biology, specifically from mesenchymal stem cell differentiation and signaling, lead to new approaches in bone regeneration. Novel scaffold and drug release materials based on polysaccharides gain increasing attention due to their wide availability and good biocompatibility to be used as hydrogels and/or hybrid components for drug release and tissue engineering. This article reviews the current state of the art, recent developments, and future perspectives in polysaccharide-based systems used for bone regeneration.

Differentiation potential of adult human mesenchymal stem cells.

Human mesenchymal stem cells (HMSCs) which are isolated from bone marrow stroma, peripheral blood, dermis, muscle and adipose tissue have the advantage of potential autologous transplantation ability. They can be differentiated into chondrogenic, osteogenic, adipogenic and myogenic lineages. Problems of stem cells from bone marrow are low cell numbers, low isolated volumes, pain, and to some extent ethical concerns. The isolation of mesenchymal stem cells from human adipose tissue was recently identified as an alternative source, since these cells are easy to obtain in big cell numbers. Adipose tissue is derived from embryonic mesoderm and contains a heterogeneous stromal cell population. To achieve lineage-specific differentiation of these cells they have to be cultured in media supplemented with appropriate factors. Inductions of the cells into multiple mesenchymal lineages resulted in the expression of several lineage-specific genes, proteins and specific metabolic activity. In conclusion, the potential benefit of the multi-germline capacity of HMSCs seems to be a promising approach for allogenic cell therapy and human tissue engineering.

Template-Mediated Biomineralization for Bone Tissue Engineering.

Template-mediated mineralization describes a research field of materials chemistry that deals with templates influencing product formation of foremost inorganic functional materials and composites. These templates are usually organic compounds - as far as molecules with natural origin are involved, the terminology "biomineralization" or "biomimetic mineralization: is used. The present review gives insight into recent developments in the research area of bone-tissue engineering with focus on chemical templates and cell-based approaches. The review is structured as follows: (1) a brief general overview about the principle of templating and recently used template materials, (2) important analytical methods, (3) examples of template-guided mineralization of various bone-related materials, (4) natural bone mineralization, (5) scaffolds for bone-tissue regeneration and (6) cell-based therapeutic approaches. For this purpose, a literature screening with emphasis on promising potential practical applications was performed. In particular, macromolecular structures and polymer composites with relation to naturally occurring compounds were favored. Priority was given to publications of the last five years. Although the present review does not cover the whole topic to full extent, it should provide information about current trends and the most promising approaches in the research area of bone-tissue engineering based on applications of organic templates/scaffolds as well as cell-based strategies.

Mesenchymal Stem Cell Differentiation into Adipocytes Is Equally Induced by Insulin and Proinsulin In Vitro.

BACKGROUND AND OBJECTIVES: In advanced ß-cell dysfunction, proinsulin is increasingly replacing insulin as major component of the secretion product. It has been speculated that proinsulin has at least the same adipogenic potency than insulin, leading to an increased tendency of lipid tissue formation in patients with late stage ß-cell dysfunction. METHODS AND RESULTS: Mesenchymal stem cells obtained from liposuction material were grown in differentiation media containing insulin (0.01 µmol), proinsulin (0.01 µmol) or insulin+proinsulin (each 0.005 µmol). Cell culture supernatants were taken from these experiments and an untreated control at weeks 1, 2, and 3, and were stored at -80°C until analysis. Cell differentiation was microscopically supervised and adiponectin concentrations were measured as marker for differentiation into mature lipid cells. This experiment was repeated three times. No growth of lipid cells and no change in adiponectin values was observed in the negative control group (after 7/14/12 days: 3.2±0.5/3.3±0.1/4.4±0.5 ng/ml/12 h). A continuous differentiation into mature adipocytes (also confirmed by Red-Oil-staining) and a corresponding increase in adiponectin values was observed in the experiments with insulin (3.6±1.9/5.1±1.4/13.3±1.5 ng/ml/12 h; p<0.05 week 1 vs. week 3) and proinsulin (3.3±1.2/3.5±0.3/12.2±1.2 ng/ml/12 h; p<0.05). Comparable effects were seen with the insulin/proinsulin combination. CONCLUSIONS: Proinsulin has the same adipogenic potential than insulin in vitro. Proinsulin has only 10∼20% of the glucose-lowering effect of insulin. It can be speculated that the adipogenic potential of proinsulin may be a large contributor to the increased body weight problems in patients with type 2 diabetes and advanced ß-cell dysfunction.

Carbon monoxide protects pancreatic beta-cells from apoptosis and improves islet function/survival after transplantation.

Pancreatic islets transplanted to treat autoimmune type 1 diabetes often fail to function (primary nonfunction), likely because of islet beta-cell apoptosis. We show that carbon monoxide (CO), a product of heme oxygenase activity, protects beta-cells from apoptosis. Protection is mediated through guanylate cyclase activation, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinases. This antiapoptotic effect is still observed when beta-cells are exposed to CO for 1 h before the apoptotic stimulus. In a similar manner, mouse islets exposed to CO for just 2 h function significantly better after transplantation than islets not exposed to CO. These findings suggest a potential therapeutic application for CO in improving islet function/survival after transplantation in humans.

The role of purinergic receptors in stem cell differentiation.

A major challenge modern society has to face is the increasing need for tissue regeneration due to degenerative diseases or tumors, but also accidents or warlike conflicts. There is great hope that stem cell-based therapies might improve current treatments of cardiovascular diseases, osteochondral defects or nerve injury due to the unique properties of stem cells such as their self-renewal and differentiation potential. Since embryonic stem cells raise severe ethical concerns and are prone to teratoma formation, adult stem cells are still in the focus of research. Emphasis is placed on cellular signaling within these cells and in between them for a better understanding of the complex processes regulating stem cell fate. One of the oldest signaling systems is based on nucleotides as ligands for purinergic receptors playing an important role in a huge variety of cellular processes such as proliferation, migration and differentiation. Besides their natural ligands, several artificial agonists and antagonists have been identified for P1 and P2 receptors and are already used as drugs. This review outlines purinergic receptor expression and signaling in stem cells metabolism. We will briefly describe current findings in embryonic and induced pluripotent stem cells as well as in cancer-, hematopoietic-, and neural crest-derived stem cells. The major focus will be placed on recent findings of purinergic signaling in mesenchymal stem cells addressed in in vitro and in vivo studies, since stem cell fate might be manipulated by this system guiding differentiation towards the desired lineage in the future.