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Cellular dynamics are governed by the geometrical, mechanical and biochemical properties of the extracellular matrix (ECM) and vice versa. This reciprocity proves essential for tissue and organ viability and many forms of interactions have extensively been investigated in 2D environments over the last decades. However, with the addition of the third dimension, cellular dynamics become increasingly complex and cellular behavior in 3D environments can differ significantly from 2D. Therefore, 3D in vitro models are being developed mimicking both healthy and pathological cell niches with the highest biological conformity including high resolution of biochemical and mechanical cues to achieve further insights in cell-ECM interactions including proliferation, differentiation and migration. Furthermore, by integrating an interveined micro vasculatory system can enable the nutrient supply well as the distinct distribution of additional biochemical factors throughout ever larger tissues models. The aim of this cumulative doctoral thesis was to improve precision 3D printed cell scaffolds mimicking native tissues in geometry, composition and mechanics. In particular, the fabrication of (i) up to mm-sized scaffolds with micrometer resolution printed from gelatin derived photo-resins which are suitable to culture and analyze a variety of cell lines as well as primary cells, (ii) with defined biomechanical properties, (iii) using biomimetic 3D print templates mimicking alveolar tissue and (iv) with integrated channels for selective perfusion was investigated. Two-photon stereolithography (TPS) was adapted to fabricate up to mm-sized high-precision cell scaffolds at sub-cellular resolution from protein based photo-resins including gelatin methacrylate and bovine serum albumin and demonstrated compatibility with a variety of cells types including primary human lung fibroblasts. By tailoring the manufacturing settings such as objective specifications or laser power as well as by app, Die Zelldynamik wird von den geometrischen, mechanischen und biochemischen Eigenschaften der extrazellulären Matrix bestimmt, wobei diese wiederum von der Zelldynamik beeinflusst werden. Diese Wechselwirkung ist für die Vitalität von Geweben und Organen von entscheidender Bedeutung. Viele Formen der Zell-ECM Interaktionen wurden in den letzten Jahrzehnten eingehend in 2D Umgebungen untersucht. Durch die Einführung der dritten Dimension wird die Zelldynamik jedoch zunehmend komplexer, und das Zellverhalten in 3D Umgebungen kann sich erheblich von dem in 2D unterscheiden. Daher werden derzeit 3D-in-vitro-Modelle entwickelt, die sowohl gesunde als auch pathologische Zellnischen mit höchster biologischer Konformität nachahmen. Diese beinhalten eine hohe räumliche Auflösung biochemischer und mechanischer Signale, um weitere Einblicke in die Zell-ECM Interaktionen, einschließlich Proliferation, Differenzierung und Migration, zu gewinnen. Darüber hinaus kann durch die Integration eines vernetzten mikrovaskulären Systems die Nährstoffversorgung sowie die selektive Verteilung zusätzlicher biochemischer Faktoren in immer größeren Gewebemodellen ermöglicht werden. Das Ziel dieser kumulativen Doktorarbeit war die Verbesserung von präzisen 3D-gedruckten Zellgerüsten, in Hinblick auf die Nachahmung von nativem Gewebe in Geometrie, biochemische Zusammensetzung und Mechanik. Insbesondere wurde zur Herstellung von (i) bis zu mm-großen Gerüsten bei gleichzeitiger Auflösung im einstelligen Mikrometerbereich beigetragen. Diese Zellgerüststrukturen wurden aus Gelatine-Phototinte gedruckt und eignen sich für die Kultivierung und Analyse einer Vielzahl von Zelllinien, darunter auch Primärzellen. Mit (ii) definierten biomechanischen Eigenschaften, (iii) unter Verwendung biomimetischer 3D-Druckvorlagen und (iv) mit integrierten Kanälen für die selektive Perfusion können diese Zellgerüste Alveolargewebe in diesen Aspekten imitieren. Die Zwei-Photonen-Stereolithographie wurde adaptiert, um ho

The desmoplastic reaction observed in many cancers is a hallmark of disease progression and prognosis, particularly in breast and pancreatic cancer. Stromal-derived extracellular matrix (ECM) is significantly altered in desmoplasia, and as such plays a critical role in driving cancer progression. Using fibroblast-derived matrices (FDMs), we show that cancer cells have increased growth on cancer associated FDMs, when compared to FDMs derived from non-malignant tissue (normal) fibroblasts. We assess the changes in ECM characteristics from normal to cancer-associated stroma at the primary tumor site. Compositional, structural, and mechanical analyses reveal significant differences, with an increase in abundance of core ECM proteins, coupled with an increase in stiffness and density in cancer-associated FDMs. From compositional changes of FDM, we derived a 36-ECM protein signature, which we show matches in large part with the changes in pancreatic ductal adenocarcinoma (PDAC) tumor and metastases progression. Additionally, this signature also matches at the transcriptomic level in multiple cancer types in patients, prognostic of their survival. Together, our results show relevance of FDMs for cancer modelling and identification of desmoplastic ECM components for further mechanistic studies.

With its high-sensitivity, non-intrusive, and contactless measurement characteristics, laser-Doppler vibrometry (LDV) allows for a detailed insight into the vibrational dynamics of systems, regardless of their fragility and structural characteristics. This exploratory study investigates if and how LDV can be used for the mechanical characterization of transparent biological materials, such as hydrogels, living cells, and cellular aggregates, as well as native and engineered tissues., Die Laser-Doppler Vibrometrie (LDV) ermöglicht mit ihren hochempfindlichen, zerstörungsfreien und kontaktlosen Messeigenschaften einen detaillierten Einblick in die Schwingungsdynamik technischer Systeme, unabhängig von deren Fragilität und strukturellen Eigenschaften. Diese explorative Studie untersucht, ob und wie LDV für die mechanische Charakterisierung von transparenten biologischen Materialien wie Hydrogelen, lebenden Zellen sowie nativen und künstlichen Geweben eingesetzt werden kann.

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNASeq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.

With its high-sensitivity, non-intrusive, and contactless measurement characteristics, laser-Doppler vibrometry (LDV) allows for a detailed insight into the vibrational dynamics of systems, regardless of their fragility and structural characteristics. This exploratory study investigates if and how LDV can be used for the mechanical characterization of transparent biological materials, such as hydrogels, living cells, and cellular aggregates, as well as native and engineered tissues., Die Laser-Doppler Vibrometrie (LDV) ermöglicht mit ihren hochempfindlichen, zerstörungsfreien und kontaktlosen Messeigenschaften einen detaillierten Einblick in die Schwingungsdynamik technischer Systeme, unabhängig von deren Fragilität und strukturellen Eigenschaften. Diese explorative Studie untersucht, ob und wie LDV für die mechanische Charakterisierung von transparenten biologischen Materialien wie Hydrogelen, lebenden Zellen sowie nativen und künstlichen Geweben eingesetzt werden kann.

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4(-/-) mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4(-/-) mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4(-/-) mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4(-/-) mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone.

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone.