Entwicklung der Hauptkomponenten eines Gelenks zur in vitro Simulation von Arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases (prevalence 0.5-1.0%), which can lead to pain and a considerable loss of life quality in affected patients. Many details of underlying causes and mechanisms are still elusive. The persistent autoimmune-mediated inflammation of the joint is one of the key features of this systemic, chronic-inflammatory disease accounting for progressive cartilage destruction. Despite major progress in the treatment of RA, a strong unmet medical need remains. Therefore, a better understanding of the underlying pathomechanisms driving RA progression is required to develop new therapeutic strategies to effectively treat patients at every stage of disease progression. Although various RA models already exist, they either employ phylogenetically distant species or rely on human cells cultured in an oversimplified environment. To date, none of these models allows sufficient or complete extrapolation to the human patient. Therefore, the use of human-based in vitro 3D tissue equivalents of an artificial joint is a promising alternative approach to investigate pathomechanisms and test new therapeutic approaches. Hence, the aim of this thesis was to develop and characterize human in vitro 3D tissue equivalents of the joint, namely (i) cancellous bone, (ii) articular cartilage, and (iii) synovial membrane using bone marrow-derived mesenchymal stromal cells (MSCs). Here, MSCs provide the possibility of producing a complete joint model from single donor material thus offering the opportunity of a personalized testing platform. The results described in this thesis show the potential of mimicking key features of arthritis in three different tissues of a joint. Firstly, to simulate the bone component, β-tricalcium phosphate (TCP) – mimicking the mineral bony part – was populated with MSCs. Cell seeding was optimized using cell sheet technology. In contrast, the cartilage component was produced by cellular self-assembly without any supporting materials. Both models exhibit phenotypic features of native tissue, including expression of bone- or cartilage-related markers, mineralization of bone that was absent in cartilage, and development of distinct zones in the glycosaminoglycan-rich cartilage model. Co-cultivation of both tissue models generated the osteochondral unit characterized by inter-tissue connectivity, cell colonization, and initial calcification implying a functional transitional bridging area. Finally, the synovial membrane model was generated based on a xeno-free synthetic hydrogel populated with MSCs indistinguishable from synovial fibroblasts with regard to classical markers. Similar to the human synovial membrane, a confluent layer of up to four cell layers was detectable within the hydrogel allowing immune cell migration. To simulate inflammation, the individual tissue components were treated with cytokines relevant for RA, such as interleukin-6, tumor necrosis factor-α, and macrophage migration inhibitory factor. This resulted in cytokine-driven cell- and matrix-related changes in accordance with those observed during RA, while treatment using biologics prevented the induction of arthritis in the osteochondral model. These results confirm the pathophysiological mutability, architecture, integrity, and viability of the distinct in vitro 3D joint components. Prospectively, the complete in vitro 3D joint model will serve as a preclinical test platform in basic and applied biomedical research to (i) study pathophysiological processes of musculoskeletal diseases, (ii) identify new potential targets, (iii) test novel therapeutic strategies, including biologic therapies, and (iv) reduce the number of animal experiments.Rheumatoide Arthritis (RA) ist eine der häufigsten Autoimmunerkrankungen (Prävalenz 0,5-1,0 %), die bei den betroffenen Patienten zu Schmerzen und einem erheblichen Verlust an Lebensqualität führen kann. Zugrundeliegende Ursachen und Mechanismen sind nach wie vor ungeklärt. Die anhaltende autoimmunvermittelte Entzündung des Gelenks ist eines der Hauptmerkmale dieser systemisch, chronisch-entzündlichen Gelenkerkrankung. Trotz erheblicher Fortschritte bei der Behandlung der Betroffenen besteht nach wie vor eine große medizinische Versorgungslücke. Ein besseres Verständnis der zugrundeliegenden Pathomechanismen ist daher die Grundvoraussetzung für die Entwicklung neuer Therapieansätze. Bisher wurden zahlreiche RA-Modelle entwickelt, jedoch basieren diese entweder auf phylogenetisch unterschiedlichen Spezies oder auf stark vereinfachten humanen in vitro Kulturen. Bis heute lassen sich die Ergebnisse aus diesen Modellen nicht ausreichend oder vollständig auf den Patienten extrapolieren. Die Verwendung von humanen in vitro 3D Gewebeäquivalenten des Gelenks ist daher ein vielversprechender präklinischer Ansatz zur Untersuchung von Pathomechanismen und zur Prüfung neuer Therapien. Ziel dieser Arbeit war es, basierend auf humanen mesenchymalen Stromazellen (MSCs), in vitro 3D Gewebeäquivalente des Gelenks zu entwickeln. Diese Gewebeäquivalente umfassen (i) spongiösen Knochen, (ii) Gelenkknorpel und (iii) Synovialmembran. MSCs bieten hierbei die Möglichkeit, ein Gelenkmodell ausgehend vom Material eines einzelnen Spenders zu entwickeln. Das 3D Knochenmodell basiert auf dem Trägermaterial β-Trikalziumphosphat (TCP), das den mineralischen Knochenanteil des Gelenkes simuliert. Die Aussaat von Zellen auf das Trägermaterial TCP wurde mittels ‚sheet technology‘ optimiert. Im Gegensatz dazu wurde das 3D Knorpelmodell durch zelluläre Selbstorganisation, frei von zusätzlichen Trägermaterialen erzeugt. Beide Modelle weisen dem nativen Gewebe ähnelnde phänotypische Merkmale auf, wie z.B. die Expression von Knochen- oder Knorpel-spezifischen Markern, die Mineralisierung des Knochens, die im Knorpel fehlte, und die Entwicklung charakteristischer Zonen des Glykosaminoglykan-reichen Knorpelmodells. Zudem generierte die Co-Kultivierung beider Modelle eine osteochondrale Einheit, die durch Kolonisierung, Konnektivität und initiale Kalzifizierung gekennzeichnet war. Diese Eigenschaften deuten auf einen funktionellen Übergangsbereich beider Gewebeäquivalente hin. Schließlich wurde das xeno-freie Synovialmembranmodell basierend auf einem synthetischen Hydrogel entwickelt. Da sowohl MSCs als auch synovialen Fibroblasten anhand klassischer Marker nicht unterscheidbar sind, wurden MSCs in das Hydrogel eingebracht. Das Synovialmembranmodell wies eine konfluente Schicht von bis zu vier Zellschichten auf, was der physiologischen Struktur der humanen Synovialmembran entspricht. Das Hydrogel ermöglicht zudem die Migration von Immunzellen; die Voraussetzung für die Simulation des inflammatorischen Zustandes. Final wurden RA-relevante Zytokine wie Interleukin-6, Tumornekrosefaktor-α und Makrophagen-Migrationshemmungsfaktor appliziert, um Entzündungsprozesse in den Gewebeäquivalenten zu simulieren. Die Zytokin-vermittelte Stimulation resultierte in Zell- und Matrix-Veränderungen, wie sie auch bei RA beobachtet werden können. Die therapeutische Intervention hingegen führte zur Reduktion der Ausprägung der Arthritis im osteochondralen Modell. Diese Ergebnisse bestätigen die pathophysiologische Variabilität, Architektur, Integrität und Lebensfähigkeit der verschiedenen in vitro 3D Gelenkkomponenten. Perspektivisch soll das kombinierte in vitro 3D Gelenkmodell als präklinische Testplattform in der Grundlagenforschung und angewandten biomedizinischen Forschung dienen, um schlussendlich (i) pathophysiologische Prozesse muskuloskelettaler Erkrankungen studieren zu können, (ii) neue potenzielle Zielmoleküle zu identifizieren, (iii) neue therapeutische Strategien einschließlich Therapien mit Biologika zu testen und (iv) Tierversuche zu reduzieren

Modeling Rheumatoid Arthritis In Vitro: From Experimental Feasibility to Physiological Proximity

Rheumatoid arthritis (RA) is a chronic, inflammatory, and systemic autoimmune disease that affects the connective tissue and primarily the joints. If not treated, RA ultimately leads to progressive cartilage and bone degeneration. The etiology of the pathogenesis of RA is unknown, demonstrating heterogeneity in its clinical presentation, and is associated with autoantibodies directed against modified self-epitopes. Although many models already exist for RA for preclinical research, many current model systems of arthritis have limited predictive value because they are either based on animals of phylogenetically distant origin or suffer from overly simplified in vitro culture conditions. These limitations pose considerable challenges for preclinical research and therefore clinical translation. Thus, a sophisticated experimental human-based in vitro approach mimicking RA is essential to (i) investigate key mechanisms in the pathogenesis of human RA, (ii) identify targets for new therapeutic approaches, (iii) test these approaches, (iv) facilitate the clinical transferability of results, and (v) reduce the use of laboratory animals. Here, we summarize the most commonly used in vitro models of RA and discuss their experimental feasibility and physiological proximity to the pathophysiology of human RA to highlight new human-based avenues in RA research to increase our knowledge on human pathophysiology and develop effective targeted therapies

Optimization of a Tricalcium Phosphate-Based Bone Model Using Cell-Sheet Technology to Simulate Bone Disorders

Bone diseases such as osteoporosis, delayed or impaired bone healing, and osteoarthritis still represent a social, financial, and personal burden for affected patients and society. Fully humanized in vitro 3D models of cancellous bone tissue are needed to develop new treatment strategies and meet patient-specific needs. Here, we demonstrate a successful cell-sheet-based process for optimized mesenchymal stromal cell (MSC) seeding on a beta-tricalcium phosphate (TCP) scaffold to generate 3D models of cancellous bone tissue. Therefore, we seeded MSCs onto the beta-TCP scaffold, induced osteogenic differentiation, and wrapped a single osteogenically induced MSC sheet around the pre-seeded scaffold. Comparing the wrapped with an unwrapped scaffold, we did not detect any differences in cell viability and structural integrity but a higher cell seeding rate with osteoid-like granular structures, an indicator of enhanced calcification. Finally, gene expression analysis showed a reduction in chondrogenic and adipogenic markers, but an increase in osteogenic markers in MSCs seeded on wrapped scaffolds. We conclude from these data that additional wrapping of pre-seeded scaffolds will provide a local niche that enhances osteogenic differentiation while repressing chondrogenic and adipogenic differentiation. This approach will eventually lead to optimized preclinical in vitro 3D models of cancellous bone tissue to develop new treatment strategies

JAK/STAT Activation: A General Mechanism for Bone Development, Homeostasis, and Regeneration

The Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling pathway serves as an important downstream mediator for a variety of cytokines, hormones, and growth factors. Emerging evidence suggests JAK/STAT signaling pathway plays an important role in bone development, metabolism, and healing. In this light, pro-inflammatory cytokines are now clearly implicated in these processes as they can perturb normal bone remodeling through their action on osteoclasts and osteoblasts at both intra- and extra-articular skeletal sites. Here, we summarize the role of JAK/STAT pathway on development, homeostasis, and regeneration based on skeletal phenotype of individual JAK and STAT gene knockout models and selective inhibition of components of the JAK/STAT signaling including influences of JAK inhibition in osteoclasts, osteoblasts, and osteocytes

Impact of Janus Kinase Inhibition with Tofacitinib on Fundamental Processes of Bone Healing

Both inflammatory diseases like rheumatoid arthritis (RA) and anti-inflammatory treatment of RA with glucocorticoids (GCs) or non-steroidal anti-inflammatory drugs (NSAIDs) negatively influence bone metabolism and fracture healing. Janus kinase (JAK) inhibition with tofacitinib has been demonstrated to act as a potent anti-inflammatory therapeutic agent in the treatment of RA, but its impact on the fundamental processes of bone regeneration is currently controversially discussed and at least in part elusive. Therefore, in this study, we aimed to examine the effects of tofacitinib on processes of bone healing focusing on recruitment of human mesenchymal stromal cells (hMSCs) into the inflammatory microenvironment of the fracture gap, chondrogenesis, osteogenesis and osteoclastogenesis. We performed our analyses under conditions of reduced oxygen availability in order to mimic the in vivo situation of the fracture gap most optimal. We demonstrate that tofacitinib dose-dependently promotes the recruitment of hMSCs under hypoxia but inhibits recruitment of hMSCs under normoxia. With regard to the chondrogenic differentiation of hMSCs, we demonstrate that tofacitinib does not inhibit survival at therapeutically relevant doses of 10-100 nM. Moreover, tofacitinib dose-dependently enhances osteogenic differentiation of hMSCs and reduces osteoclast differentiation and activity. We conclude from our data that tofacitinib may influence bone healing by promotion of hMSC recruitment into the hypoxic microenvironment of the fracture gap but does not interfere with the cartilaginous phase of the soft callus phase of fracture healing process. We assume that tofacitinib may promote bone formation and reduce bone resorption, which could in part explain the positive impact of tofacitinib on bone erosions in RA. Thus, we hypothesize that it will be unnecessary to stop this medication in case of fracture and suggest that positive effects on osteoporosis are likely

Production of IL-6 and Phagocytosis Are the Most Resilient Immune Functions in Metabolically Compromised Human Monocytes

At sites of inflammation, monocytes carry out specific immune functions while facing challenging metabolic restrictions. Here, we investigated the potential of human monocytes to adapt to conditions of gradually inhibited oxidative phosphorylation (OXPHOS) under glucose free conditions. We used myxothiazol, an inhibitor of mitochondrial respiration, to adjust two different levels of decreased mitochondrial ATP production. At these levels, and compared to uninhibited OXPHOS, we assessed phagocytosis, production of reactive oxygen species (ROS) through NADPH oxidase (NOX), expression of surface activation markers CD16, CD80, CD11b, HLA-DR, and production of the inflammatory cytokines IL-1 beta, IL-6 and TNF-alpha in human monocytes. We found phagocytosis and the production of IL-6 to be least sensitive to metabolic restrictions while surface expression of CD11b, HLA-DR, production of TNF-alpha, IL-1 beta and production of ROS through NOX were most compromised by inhibition of OXPHOS in the absence of glucose. Our data demonstrate a short-term hierarchy of immune functions in human monocytes, which represents novel knowledge potentially leading to the development of new therapeutics in monocyte-mediated inflammatory diseases

Functional Scaffold‐Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (mu CT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1 beta were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. (c) 2021 American Society for Bone and Mineral Research (ASBMR)

Macroscale mesenchymal condensation to study cytokine-driven cellular and matrix-related changes during cartilage degradation

Understanding the pathophysiological processes of cartilage degradation requires adequate model systems to develop therapeutic strategies towards osteoarthritis (OA). Although different in vitro or in vivo models have been described, further comprehensive approaches are needed to study specific disease aspects. This study aimed to combine in vitro and in silico modeling based on a tissue-engineering approach using mesenchymal condensation to mimic cytokine-induced cellular and matrix-related changes during cartilage degradation. Thus, scaffold-free cartilage-like constructs (SFCCs) were produced based on self-organization of mesenchymal stromal cells (mesenchymal condensation) and (i) characterized regarding their cellular and matrix composition or secondly (ii) treated with interleukin-1β (IL–1β) and tumor necrosis factor α (TNFα) for 3 weeks to simulate OA-related matrix degradation. In addition, an existing mathematical model based on partial differential equations was optimized and transferred to the underlying settings to simulate the distribution of IL–1β, type II collagen degradation and cell number reduction. By combining in vitro and in silico methods, we aimed to develop a valid, efficient alternative approach to examine and predict disease progression and effects of new therapeutics.publishedVersio

Local immune cell contributions to fracture healing in aged individuals – A novel role for interleukin 22

Aging: immune protein's role in delayed bone fracture healing Neutralizing a key cytokine, a signaling protein affecting the immune system could rejuvenate the healing process following prolonged inflammatory responses to bone fractures in elderly patients. Healing patterns vary widely in the elderly following injuries such as bone fractures, and scientists now believe that a patient's individual innate and adaptive immune profile directly affects the healing process. A short-lived pro-inflammatory response is needed to kickstart healthy healing, but a longer-lasting response can be damaging. In experiments on aged mouse models, the team led by Katharina Schmidt-Bleek at the Julius Wolff Institute in Berlin, Germany, demonstrated that high levels of the cytokine interleukin-22 impaired bone regeneration. Elevated interleukin-22 levels resulted from chronically elevated inflammation and inflammaging, prevalent in elderly patients. The team treated the mice to neutralize interleukin-22, which accelerated the healing process. With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences

Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

Cellular metabolism modulates effector functions in human CD4+ T (Th) cells by providing energy and building blocks. Conversely, cellular metabolic responses are modulated by various influences, e.g., age. Thus, we hypothesized that metabolic reprogramming in human Th cells during aging modulates effector functions and contributes to “inflammaging”, an aging-related, chronic, sterile, low-grade inflammatory state characterized by specific proinflammatory cytokines. Analyzing the metabolic response of human naive and memory Th cells from young and aged individuals, we observed that memory Th cells exhibit higher glycolytic and mitochondrial fluxes than naive Th cells. In contrast, the metabolism of the latter was not affected by donor age. Memory Th cells from aged donors showed a higher respiratory capacity, mitochondrial content, and intracellular ROS production than those from young donors without altering glucose uptake and cellular ATP levels, which finally resulted in higher secreted amounts of proinflammatory cytokines, e.g., IFN-γ, IP-10 from memory Th cells taken from aged donors after TCR-stimulation which were sensitive to ROS inhibition. These findings suggest that metabolic reprogramming in human memory Th cells during aging results in an increased expression of proinflammatory cytokines through enhanced ROS production, which may contribute to the pathogenesis of inflammaging