Skeletal bone function relies on both cells and cellular niches, which, when combined, provide guiding cues for the control of differentiation and remodeling processes. Here, we propose an in vitro 3D model based on human fetal osteoblasts, which eases the study of osteocyte commitment in vitro and thus provides a means to examine the influences of biomaterials, substances or cells on the regulation of these processes. Aggregates were formed from human fetal osteoblasts (hFOB1.19) and cultivated under proliferative, adipo- and osteoinductive conditions. When cultivated under osteoinductive conditions, the vitality of the aggregates was compromised, the expression levels of the mineralization-related gene DMP1 and the amount of calcification and matrix deposition were lower, and the growth of the spheroids stalled. However, within spheres under growth conditions without specific supplements, self-organization processes occur, which promote extracellular calcium deposition, and osteocyte-like cells develop. Long-term cultivated hFOB aggregates were free of necrotic areas. Moreover, hFOB aggregates cultivated under standard proliferative conditions supported the co-cultivation of human monocytes, microvascular endothelial cells and stromal cells. Overall, the model presented here comprises a self-organizing and easily accessible 3D osteoblast model for studying bone marrow formation and in vitro remodeling and thus provides a means to test druggable molecular pathways with the potential to promote life-long bone formation and remodeling.

The wide use of 3D-organotypic cell models is imperative for advancing our understanding of basic cell biological mechanisms. For this purpose, easy-to-use enabling technology is required, which should optimally link standardized assessment methods to those used for the formation, cultivation, and evaluation of cell aggregates or primordial tissue. We thus conceived, manufactured, and tested devices which provide the means for cell aggregation and online monitoring within a hanging drop. We then established a workflow for spheroid manipulation and immune phenotyping. This described workflow conserves media and reagent, facilitates the uninterrupted tracking of spheroid formation under various conditions, and enables 3D-marker analysis by means of 3D epifluorescence deconvolution microscopy. We provide a full description of the low-cost manufacturing process for the fluidic devices and microscopic assessment tools, and the detailed blueprints and building instructions are disclosed. Conclusively, the presented compilation of methods and techniques promotes a quick and barrier-free entry into 3D cell biology.

Advancements in biomaterial manufacturing technologies calls for improved standards of fabrication and testing. Currently 3D-printable resins are being formulated which exhibit the potential to rapidly prototype biocompatible devices. For validation purposes, 3D-printed materials were subjected to a hierarchical validation onto the chorioallantoic membrane of the developing chicken, better known as the HET CAM assay. Working along these lines, prints made from poly-(ethylene glycol)-diacrylate (PEGDA), which had undergone appropriate post-print processing, outperformed other commercial resins. This material passed all tests without displaying adverse effects, as experienced with other resin types. Based on this finding, the micro bioreactors (MBR) design, first made of PDMS and that also passed with cell tests on the HET-CAM, was finally printed in PEGDA, and applied in vivo. Following this workflow shows the applicability of 3D-printable resins for biomedical device manufacturing, consents to adherence to the present standards of the 3R criteria in material research and development, and provides flexibility and fast iteration of design and test cycles for MBR adaptation and optimization.

The human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

Background: Due to aging, tissue regeneration gradually declines. Contemporary strategies to promote tissue-specific regeneration, in particular in elderly patients, often include synthetic material apt for implantation primarily aiming at upholding body functions and regaining appropriate anatomical and functional integrity. Objective: Biomaterials suitable for complex reconstruction surgical procedures have to exert high physicochemical stability and biocompatibility. Method: A polymer made of poly-L-lactic acid and poly-ε-caprolactone was synthesized by means of a novel tin-free catalytic process. The material was tested in a bioreactor-assisted perfusion culture and implanted in a sheep model for lateral augmentation of the mandible. Histological and volumetric evaluation was performed 3 and 6 months post-implantation. Results: After synthesis the material could be further refined by cryogrinding and sintering, thus yielding differently porous scaffolds that exhibited a firm and stable appearance. In perfusion culture, no disintegration was observed for extended periods of up to 7 weeks, while mesenchymal stromal cells readily attached to the material, steadily proliferated, and deposited extracellular calcium. The material was tested in vivo together with autologous bone marrow-derived stromal cells. Up to 6 months post-implantation, the material hardly changed in shape with composition also refraining from foreign body reactions. Conclusion: Given the long-term shape stability in vivo, featuring imperceptible degradation and little scarring as well as exerting good compatibility to cells and surrounding tissues, this novel biomaterial is suitable as a space filler in large anatomical defects.

Background: Bone ageing is governed by the linked activities of short-lived osteoblasts and osteoclasts in conjunction with long-lived osteocytes present in osseous structure. Besides their maintenance function, osteogenic cells also gain specific positional information, which may potentially trigger ageing-associated cellular deviations in terminally differentiated osteocytes differently in cranial versus postcranial tissues. Methods: We therefore investigated bone taken from deceased aged humans explanted at five distinct anatomical positions throughout the body and assessed physical and biological determinants applying radiologic and histologic measures. Results: We were able to show that significantly more osteocytes reside in aged cortical bone at cranial positions than within axial or limb skeleton. These cellular states and conditions were not found in the corresponding trabecular bone, where osteocyte numbers remain also high at postcranial positions. Parallel comparative analyses of bone microstructure as analyzed by means of computer tomography showed no significant differences. Conclusions: Considering differences and commonalities regarding the bone samples, such as loading, mechanisms of ossification or the surrounding stromal cell compartment, our findings indicate that positional information laid down during ontogenetic processes is instructive during the entire life thus potentially also moulding spatial-specific mechanistic distinctions of bone ageing. (C) 2019 Elsevier GmbH. All rights reserved.

Nanoparticles, especially from carbon, have great potential in biomedicine. However, prior to clinical use, biocompatibility and biodistribution of these nanoobjects have to be assessed. Currently, particle detection is mostly based on surface‐bound labels, inevitably altering materials' properties by surface modification. Further obstacles include bleaching, dissociation of labels from the surface, weak emission of fluorophores due to insufficient tissue opacity or hampered light penetration or the need for specific excitation wavelengths. These characteristics greatly constrain employment of such nanoparticles to address complex analytical questions. To overcome these drawbacks, the use of intrinsic structural features of nanoobjects is highly desirable: the particle surface remains unchanged and the nanoobject exhibits its innate behavior. Thus, for sensitive detection and quantification, labels should be incorporated in the nanoparticle core, thereby avoiding cleavage and warranting unchanged surface characteristics. The incorporation of clinically approved radionuclide 32 P into the lattice of nanodiamond particles using highly defined ion implantation is described here. The properties, uptake, and biodistribution are studied in vivo, in the developing hen's egg model (hen's egg test on chorioallantoic membrane assay). It is found that 32 P labeling of diamond nanoparticles allows their reliable localization and sensitive quantification in a cost efficient, highly reliable, and safe way using available autoradiographic devices and analytical methods.

BACKGROUND:Osteocytes are engaged in life-enduring processes such as bone remodelling, fracture healing or osseointegration of implants. Over age, ossification processes and regenerative capacity can greatly differ in mandible and femur.OBJECTIVE:Mesenchymal stem cells from cranial and postcranial bones are of different embryologic origin. This may be the reason why the regenerative capacity differs between cranial and postcranial bones in old patients. It was hypothesised that different ageing patterns, reflected by osteocyte density, lacunar density and osteoid formation, exist between murine mandibles and femurs.MATERIAL AND METHODS:Mandible and femur of young (4 months) and old (34-36 months old) male C57Bl/6 mice were histologically investigated to determine the number of lacunae occupied with osteocytes. Osteoid formation was revealed by Masson-Goldner staining, and the spatial distribution of BMP-2 synthesis was examined.RESULTS:Over lifetime, the number of lacunae occupied with osteocytes only showed a modest decrease in mandibular bone (old 85.63%/young 91.12%) while greatly diverging in the femur (old 55.99%/young 93.28%). In equal measure, old femur exhibited less osteoid formation and decreased BMP-2 expression.CONCLUSION:Tissue-specific conduct of bone ageing is moulded by osteocytic activities, which was found to vary between postcranial and craniofacial skeleton. The latter harbours long-lived osteocytes also in old animals which assures lifelong bone integrity. Preliminary concurring findings from a human cadaver, also presented in this contribution, provided a rationale for recommending the translatability to humans.

Biofunctionalization of scaffold materials can enable the healing of large bone defects. In case of minimally invasive guided-bone regeneration (GBR), limitations are however hard-to-control side effects related to the potential release of biofactors into the systemic environment. Biofactors can be stably bound to nanodiamond particles (ND) through physisorption. We therefore tested the biological and clinical effects of refining beta-tricalcium phosphate (βTCP) with ND in vitro and in vivo. In vitro, βTCP carrying 4% ND resulted in enhanced attachment of mesenchymal stem cells. When assessing GBR after lateral augmentation of the mandible in sheep showed that ND in βTCP resulted in a consistently steady bone formation when compared to pure βTCP, demonstrating the biological inert behavior and the potential clinical safety of ND.

Stem cells are considered distinctly localized within tissues because of specific anatomical structures called the stem cell niche. Intimately connected, the stem cell and the niche are subject to aging. Potent mechanisms of biological aging are control of cellular metabolism, the action of aging genes, epigenetic drift and inflammation, which also modulate the cellular age of stem cells. A common feature of aged stem cells is however hyperactivation.

Background. Radiation therapy (RT) of the head and neck region is often accompanied by serious side effects. Research in this area is needed to improve treatment outcomes and ameliorate therapy tolerance. Laboratory rodents are barely matching today's clinical standards in RT research. Yet domestic swine (Sus scrofa domestica) have previously proved suitable for various advanced tests in clinical research and training. We therefore investigated whether S. scrofa domestica is also appropriate for irradiation of the mandible.Study Design. A common scheme for irradiation treatment of S. scrofa domestica mandibles in a split-mouth design was acquired by applying computed tomography (CT) scanning under sedation. Basing on close anatomic resemblance, a standard treatment plan comprising 2 opposed irradiation fields could be accomplished.Results. RT was carried out in a clinical environment with 2 x 9 Gy. The resulting operating procedure facilitated complication-free sedation, transport, positioning, CT scanning, and effective irradiation.Conclusion. Based on common standards applied for RT in humans, domestic pigs can be employed to progress RT clinical research. Due to their human-like anatomy, physiology, size, and weight, the swine model is expedient for advancing experimental RT of the head and neck area.

Human life expectancy has increased dramatically in the last century and as a result also the prevalence of a variety of age-related diseases and syndromes. One such syndrome is frailty, which is defined as a combination of organ dysfunctions leading to increased vulnerability to adverse health outcomes. In humans, frailty is associated with various biomarkers of ageing and predicts relevant outcomes such as responses to therapies and progression of health status and mortality. Moreover, it is relatively easy to assess. To foster translation of mechanistic understanding of the ageing process and, importantly, of interventions that may extend healthy lifespan, frailty scales have been reverse translated into mice in recent years. We will review these approaches with a view to identify what is known and what is not known at present about their validity, reproducibility and reliability with a focus on the potential for further improvement.

MicroRNAs (miRNAs) are a group of small non-coding executor RNAs. Their function as key modulators of cellular senescence has been widely recognized recently. By cross-comparing several human aging models we previously identified dozens of miRNAs being differentially regulated during aging. Here the functions of two miRNAs, mir-24 and mir-424, were investigated in an oxidative stress-induced fibroblast premature senescence model. Using pre-miRNA precursors, miRNAs were overexpressed in cells undergoing premature senescence induced by oxidative stress. More senescent cells were observed in mir-24 transfected cells. p53 was upregulated in mir-24 overexpressing cells, but downregulated in mir-424 overexpressing cells. DNA topoisomerase I (TOP1), an enzyme controlling DNA topology, was identified as a target of mir-24, whose expression was induced by oxidative stress. Knocking down TOP1 induced cellular senescence. These results suggest that mir-24 activity propagates stress-induced senescence by down regulating TOP1.

Aging is associated with an accruing emergence of non-functional tissues. Mesenchymal stem cells (MSC) bring forth progenitors with multi-lineage differentiation potential, yet, they also exhibit anti-inflammatory and tissue-protective properties. Due to aging, altered tissue microenvironments constrict controlled stem cell proliferation and progenitor differentiation, thus diminishing the fitness of MSC. Therefore, deepening our understanding of metabolic, molecular and environmental factors impacting on MSC during human aging as well as providing new vistas on their role in promoting healthy aging and preventing age-associated disease is pivot. It is anticipated that integrative quantification of systemic parameters dominantly impacting on MSC will also enable effective personalized prognosis and provision of effective early medical interventions. Working along this line, it can be envisaged that standards in medical therapies can be individually adjusted by accounting not solely for the patient's chronological age or other physical parameters rather than specific physiological parameters which are believed to functionally shape stem cell niches within the bone marrow.

Do men die young and sick, or do women live long and healthy? By trying to explain the sexual dimorphism in life expectancy, both biological and environmental aspects are presently being addressed. Besides age-related changes, both the immune and the endocrine system exhibit significant sex-specific differences. This review deals with the aging immune system and its interplay with sex steroid hormones. Together, they impact on the etiopathology of many infectious diseases, which are still the major causes of morbidity and mortality in people at old age. Among men, susceptibilities toward many infectious diseases and the corresponding mortality rates are higher. Responses to various types of vaccination are often higher among women thereby also mounting stronger humoral responses. Women appear immune-privileged. The major sex steroid hormones exhibit opposing effects on cells of both the adaptive and the innate immune system: estradiol being mainly enhancing, testosterone by and large suppressive. However, levels of sex hormones change with age. At menopause transition, dropping estradiol potentially enhances immunosenescence effects posing postmenopausal women at additional, yet specific risks. Conclusively during aging, interventions, which distinctively consider the changing level of individual hormones, shall provide potent options in maintaining optimal immune functions.

The workshop entitled 'Interventions to Slow Aging in Humans: Are We Ready?' was held in Erice, Italy, on October 8-13, 2013, to bring together leading experts in the biology and genetics of aging and obtain a consensus related to the discovery and development of safe interventions to slow aging and increase healthy lifespan in humans. There was consensus that there is sufficient evidence that aging interventions will delay and prevent disease onset for many chronic conditions of adult and old age. Essential pathways have been identified, and behavioral, dietary, and pharmacologic approaches have emerged. Although many gene targets and drugs were discussed and there was not complete consensus about all interventions, the participants selected a subset of the most promising strategies that could be tested in humans for their effects on healthspan. These were: (i) dietary interventions mimicking chronic dietary restriction (periodic fasting mimicking diets, protein restriction, etc.); (ii) drugs that inhibit the growth hormone/IGF-I axis; (iii) drugs that inhibit the mTOR-S6K pathway; or (iv) drugs that activate AMPK or specific sirtuins. These choices were based in part on consistent evidence for the pro-longevity effects and ability of these interventions to prevent or delay multiple age-related diseases and improve healthspan in simple model organisms and rodents and their potential to be safe and effective in extending human healthspan. The authors of this manuscript were speakers and discussants invited to the workshop. The following summary highlights the major points addressed and the conclusions of the meeting.