Publications by authors named "Hauke Clausen-Schaumann"

54 Publications

Extending Single Cell Bioprinting from Femtosecond to Picosecond Laser Pulse Durations.

Micromachines (Basel) 2021 Sep 29;12(10). Epub 2021 Sep 29.

Lasercenter, Department of Applied Sciences and Mechatronics, Munich University of Applied Sciences, Lothstrasse 34, 80335 Munich, Germany.

Femtosecond laser pulses have been successfully used for film-free single-cell bioprinting, enabling precise and efficient selection and positioning of individual mammalian cells from a complex cell mixture (based on morphology or fluorescence) onto a 2D target substrate or a 3D pre-processed scaffold. In order to evaluate the effects of higher pulse durations on the bioprinting process, we investigated cavitation bubble and jet dynamics in the femto- and picosecond regime. By increasing the laser pulse duration from 600 fs to 14.1 ps, less energy is deposited in the hydrogel for the cavitation bubble expansion, resulting in less kinetic energy for the jet propagation with a slower jet velocity. Under appropriate conditions, single cells can be reliably transferred with a cell survival rate after transfer above 95% through the entire pulse duration range. More cost efficient and compact laser sources with pulse durations in the picosecond range could be used for film-free bioprinting and single-cell transfer.
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http://dx.doi.org/10.3390/mi12101172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8538086PMC
September 2021

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.

J Biol Chem 2021 Sep 22;297(4):101224. Epub 2021 Sep 22.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Biochemistry, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany. Electronic address:

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. scRNA-Seq 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.
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http://dx.doi.org/10.1016/j.jbc.2021.101224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8503590PMC
September 2021

Fourier Transform Infrared Microspectroscopy Combined with Principal Component Analysis and Artificial Neural Networks for the Study of the Effect of β-Hydroxy-β-Methylbutyrate (HMB) Supplementation on Articular Cartilage.

Int J Mol Sci 2021 Aug 25;22(17). Epub 2021 Aug 25.

Department of Biophysics, Faculty of Environmental Biology, University of Life Sciences in Lublin, 20-950 Lublin, Poland.

The potential of Fourier Transform infrared microspectroscopy (FTIR microspectroscopy) and multivariate analyses were applied for the classification of the frequency ranges responsible for the distribution changes of the main components of articular cartilage (AC) that occur during dietary β-hydroxy-β-methyl butyrate (HMB) supplementation. The FTIR imaging analysis of histological AC sections originating from 35-day old male piglets showed the change in the collagen and proteoglycan contents of the HMB-supplemented group compared to the control. The relative amount of collagen content in the superficial zone increased by more than 23% and in the middle zone by about 17%, while no changes in the deep zone were observed compared to the control group. Considering proteoglycans content, a significant increase was registered in the middle and deep zones, respectively; 62% and 52% compared to the control. AFM nanoindentation measurements collected from animals administered with HMB displayed an increase in AC tissue stiffness by detecting a higher value of Young's modulus in all investigated AC zones. We demonstrated that principal component analysis and artificial neural networks could be trained with spectral information to distinguish AC histological sections and the group under study accurately. This work may support the use and effectiveness of FTIR imaging combined with multivariate analyses as a quantitative alternative to traditional collagenous tissue-related histology.
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http://dx.doi.org/10.3390/ijms22179189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8430473PMC
August 2021

Basement membrane stiffness determines metastases formation.

Nat Mater 2021 06 25;20(6):892-903. Epub 2021 Jan 25.

Center for Applied Tissue Engineering and Regenerative Medicine-CANTER, Munich University of Applied Sciences, Munich, Germany.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.
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http://dx.doi.org/10.1038/s41563-020-00894-0DOI Listing
June 2021

Early Detection of Cartilage Degeneration: A Comparison of Histology, Fiber Bragg Grating-Based Micro-Indentation, and Atomic Force Microscopy-Based Nano-Indentation.

Int J Mol Sci 2020 Oct 6;21(19). Epub 2020 Oct 6.

Center for Applied Tissue Engineering and Regenerative Medicine-CANTER, Munich University of Applied Sciences, 80335 Munich, Germany.

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 µg/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 µg/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 µg/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA.
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http://dx.doi.org/10.3390/ijms21197384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582717PMC
October 2020

Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics.

Adv Healthc Mater 2020 12 7;9(24):e2000918. Epub 2020 Oct 7.

Center for NanoScience (CeNS), Ludwig-Maximilians-University, Geschwister-Scholl Platz 1, Munich, 80539, Germany.

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell-ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell-ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two-photon stereolithography is adopted to print up to mm-sized high-precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein-based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two-pass printing or post-print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7-300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D-lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single-cell and tissue dynamics in response to defined mechanical and bio-molecular cues and is ultimately scalable to full organs.
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http://dx.doi.org/10.1002/adhm.202000918DOI Listing
December 2020

Imbalanced cellular metabolism compromises cartilage homeostasis and joint function in a mouse model of mucolipidosis type III gamma.

Dis Model Mech 2020 11 18;13(11). Epub 2020 Nov 18.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany

Mucolipidosis type III (MLIII) gamma is a rare inherited lysosomal storage disorder caused by mutations in encoding the γ-subunit of GlcNAc-1-phosphotransferase, the key enzyme ensuring proper intracellular location of multiple lysosomal enzymes. Patients with MLIII gamma typically present with osteoarthritis and joint stiffness, suggesting cartilage involvement. Using knockout ( ) mice as a model of the human disease, we showed that missorting of a number of lysosomal enzymes is associated with intracellular accumulation of chondroitin sulfate in chondrocytes and their impaired differentiation, as well as with altered microstructure of the cartilage extracellular matrix (ECM). We also demonstrated distinct functional and structural properties of the Achilles tendons isolated from and knock-in ( ) mice, the latter displaying a more severe phenotype resembling mucolipidosis type II (MLII) in humans. Together with comparative analyses of joint mobility in MLII and MLIII patients, these findings provide a basis for better understanding of the molecular reasons leading to joint pathology in these patients. Our data suggest that lack of GlcNAc-1-phosphotransferase activity due to defects in the γ-subunit causes structural changes within the ECM of connective and mechanosensitive tissues, such as cartilage and tendon, and eventually results in functional joint abnormalities typically observed in MLIII gamma patients. This idea was supported by a deficit of the limb motor function in mice challenged on a rotarod under fatigue-associated conditions, suggesting that the impaired motor performance of mice was caused by fatigue and/or pain at the joint.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.046425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687858PMC
November 2020

Inadequate tissue mineralization promotes cancer cell attachment.

PLoS One 2020 28;15(8):e0237116. Epub 2020 Aug 28.

Center for Applied Tissue Engineering and Regenerative Medicine (CANTER), Munich University of Applied Sciences, Munich, Germany.

Bone metastases are a frequent complication in prostate cancer, and several studies have shown that vitamin D deficiency promotes bone metastases. However, while many studies focus on vitamin D's role in cell metabolism, the effect of chronically low vitamin D levels on bone tissue, i.e. insufficient mineralization of the tissue, has largely been ignored. To investigate, whether poor tissue mineralization promotes cancer cell attachment, we used a fluorescence based adhesion assay and single cell force spectroscopy to quantify the adhesion of two prostate cancer cell lines to well-mineralized and demineralized dentin, serving as biomimetic bone model system. Adhesion rates of bone metastases-derived PC3 cells increased significantly on demineralized dentin. Additionally, on mineralized dentin, PC3 cells adhered mainly via membrane anchored surface receptors, while on demineralized dentin, they adhered via cytoskeleton-anchored transmembrane receptors, pointing to an interaction via exposed collagen fibrils. The adhesion rate of lymph node derived LNCaP cells on the other hand is significantly lower than that of PC3 and not predominately mediated by cytoskeleton-linked receptors. This indicates that poor tissue mineralization facilitates the adhesion of invasive cancer cells by the exposure of collagen and emphasizes the disease modifying effect of sufficient vitamin D for cancer patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237116PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454967PMC
October 2020

Adhesive Properties of the Hyaluronan Pericellular Coat in Hyaluronan Synthases Overexpressing Mesenchymal Stem Cells.

Int J Mol Sci 2020 May 28;21(11). Epub 2020 May 28.

Experimental Surgery and Regenerative Medicine (ExperiMed), Department of General, Trauma and Reconstructive Surgery, Munich University Hospital, Ludwig-Maximilians-University, 80336 Munich, Germany.

Hyaluronan (HA), a natural component of the extracellular matrix, is supposed to have a regulatory function in the stem cell niche. Bone marrow-derived human mesenchymal stem cells (hMSCs) are known to express all three hyaluronan synthases (HASes), which are responsible for HA production. HA is extruded into the extracellular matrix, but also stays bound to the plasma membrane forming a pericellular coat, which plays a key role during early cell adhesion. Since HAS isoenzymes, HAS1, HAS2 and HAS3, produce HA with different molecular weights, a difference in their role for cell adhesion is expected. Here, we transduced the immortalized hMSC cell line SCP1 to constitutively express eGFP-tagged HASes (SCP1-HAS-eGFP) by lentiviral gene transfer. The overexpression of the HAS-eGFP was shown on RNA and protein levels, HA was determined by ELISA and the stained HA-coat was analyzed using confocal microscopy. Time-lapse microscopy, spreading assay and single cell force spectroscopy using atomic force microscopy were applied to characterize adhesion of the different HAS transduced SCP1 cells. We showed in this study that HAS3 overexpressing cells formed the thickest pericellular coat compared with control or HAS1 and HAS2 transduced cells. Furthermore, SCP1-HAS3-eGFP displayed faster and stronger adhesion compared to cells overexpressing the other synthases or control cells. We conclude that overexpression of HASes in hMSCs differentially modulates their initial adhesive interactions with the substrate. This observation might be helpful in regenerative medicine goals.
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http://dx.doi.org/10.3390/ijms21113827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312511PMC
May 2020

Osteoidosis leads to altered differentiation and function of osteoclasts.

J Cell Mol Med 2020 05 13;24(10):5665-5674. Epub 2020 Apr 13.

Experimental Plastic Surgery, Clinic for Plastic and Hand Surgery, Technische Universität München, Munich, Germany.

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.
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http://dx.doi.org/10.1111/jcmm.15227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214153PMC
May 2020

Loss of tenomodulin expression is a risk factor for age-related intervertebral disc degeneration.

Aging Cell 2020 03 21;19(3):e13091. Epub 2020 Feb 21.

Experimental Trauma Surgery, Department of Trauma Surgery, University Regensburg Medical Centre, Regensburg, Germany.

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti-angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament-specific marker and anti-angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age-dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd ) mice exhibited more rapid progression of age-related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD- and tendon/ligament-related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic-like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd Chm1 ) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF-derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age-related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age-related IVD degeneration.
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http://dx.doi.org/10.1111/acel.13091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059137PMC
March 2020

Three-dimensional self-assembling nanofiber matrix rejuvenates aged/degenerative human tendon stem/progenitor cells.

Biomaterials 2020 04 21;236:119802. Epub 2020 Jan 21.

Experimental Trauma Surgery, Department of Trauma Surgery, University Regensburg Medical Centre, Regensburg, Germany. Electronic address:

The poor healing capacity of tendons is known to worsen in the elderly. During tendon aging and degeneration, endogenous human tendon stem/progenitor cells (hTSPCs) experience profound pathological changes. Here, we explored a rejuvenation strategy for hTSPCs derived from aged/degenerated Achilles tendons (A-TSPCs) by providing three-dimensional (3D) nanofiber hydrogels and comparing them to young/healthy TSPCs (Y-TSPCs). RADA peptide hydrogel has a self-assembling ability, forms a nanofibrous 3D niche and can be further functionalized by adding RGD motifs. Cell survival, apoptosis, and proliferation assays demonstrated that RADA and RADA/RGD hydrogels support A-TSPCs in a comparable manner to Y-TSPCs. Moreover, they rejuvenated A-TSPCs to a phenotype similar to that of Y-TSPCs, as evidenced by restored cell morphology and cytoskeletal architecture. Transmission electron, confocal laser scanning and atomic force microscopies demonstrated comparable ultrastructure, surface roughness and elastic modulus of A- and Y-TSPC-loaded hydrogels. Lastly, quantitative PCR revealed similar expression profiles, as well a significant upregulation of genes related to tenogenesis and multipotency. Taken together, the RADA-based hydrogels exert a rejuvenating effect by recapitulating in vitro specific features of the natural microenvironment of human TSPCs, which strongly indicates their potential to direct cell behaviour and overcome the challenge of cell aging and degeneration in tendon repair.
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http://dx.doi.org/10.1016/j.biomaterials.2020.119802DOI Listing
April 2020

Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis.

Int J Mol Sci 2020 Jan 19;21(2). Epub 2020 Jan 19.

Experimental Surgery and Regenerative Medicine (ExperiMed), Department of General, Trauma and Reconstructive Surgery, Munich University Hospital, Ludwig-Maximilians-University, 80336 Munich, Germany.

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. 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 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, 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, 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.
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http://dx.doi.org/10.3390/ijms21020666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7013758PMC
January 2020

Sensory neuropeptides are required for bone and cartilage homeostasis in a murine destabilization-induced osteoarthritis model.

Bone 2020 04 8;133:115181. Epub 2020 Jan 8.

Dept. of Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology (ZMB), Bio Park 1, University of Regensburg, Germany. Electronic address:

Numerous studies identified a role for the sensory neuropeptides substance P (SP) and alpha calcitonin gene-related peptide (αCGRP) in osteoarthritis (OA) pain behavior. Surprisingly, little attention has been paid on how their trophic effects on cartilage and bone cells might affect structural changes of bone and cartilage in OA pathology. Here, we sought to elucidate sensory neuropeptides influence on structural alterations of bone and cartilage during murine OA pathophysiology. OA was induced by destabilization of the medial meniscus (DMM) in the right knee joint of 12 weeks old male C57Bl/6J wildtype (WT) mice and mice either deficient for SP (tachykinin 1 (Tac1)-/-) or αCGRP. By OARSI histopathological grading we observed significant cartilage matrix degradation after DMM surgery in αCGRP-deficient mice after 4 weeks whereas Tac1-/- scores were not different to sham mice before 12 weeks after surgery. Indentation-type atomic force microscopy (IT-AFM) identified a strong superficial zone (SZ) cartilage phenotype in Tac1-/- Sham mice. Opposed to WT and αCGRP-/- mice, SZ cartilage of Tac1-/- mice softened 2 weeks after OA induction. In Tac1-/- DMM mice, bone volume to total volume ratio (BV/TV) increased significantly compared to the Tac1-/- Sham group, 2 weeks after surgery. WT mice had reduced BV/TV compared to αCGRP-/- and Tac1-/- mice after 12 weeks. Increased calcified cartilage thickness and medial condyle diameter were detected in the medial tibia of all groups 8 weeks after OA induction by nanoCT analysis. Meniscal ossification occurred in all OA groups, but was significantly stronger in the absence of neuropeptides. Increased serum concentration of the respective non-deleted neuropeptide was observed in both neuropeptide-deficient mice strains. Both neuropeptides protect from age-related bone structural changes under physiological conditions and SP additionally demonstrates an anabolic effect on bone structure preservation in a pathophysiological situation. Both neuropeptide deficient mice display an intrinsic structural cartilage matrix phenotype that might alter progression of cartilage degeneration in OA.
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http://dx.doi.org/10.1016/j.bone.2019.115181DOI Listing
April 2020

A laser-cutting-based manufacturing process for the generation of three-dimensional scaffolds for tissue engineering using Polycaprolactone/Hydroxyapatite composite polymer.

J Tissue Eng 2019 Jan-Dec;10:2041731419859157. Epub 2019 Jul 23.

Center for Applied Tissue Engineering and Regenerative Medicine (CANTER), University of Applied Sciences Munich, Munich, Germany.

A manufacturing process for sheet-based stacked scaffolds (SSCs) based on laser-cutting (LC) was developed. The sheets consist of Polycaprolactone/Hydroxyapatite (PCL/HA) composite material. Single sheets were cut from a PCL/HA foil and stacked to scaffolds with interconnecting pores of defined sizes. HA quantities up to 50% were processable with high reproducibility, while the accuracy was dependent on the applied laser power. The smallest achievable pore sizes were about 40 µm, while the smallest stable solid structures were about 125 µm. The human mesenchymal stem cell line SCP-1 was cultured on the manufactured PCL/HA scaffolds. The cells developed a natural morphology and were able to differentiate to functional osteoblasts. The generation of PCL/HA SSCs via LC offers new possibilities for tissue engineering (TE) approaches. It is reliable and fast, with high resolution. The SSC approach allows for facile cell seeding and analysis of cell fate within the three-dimensional cell culture, thus allowing for the generation of functional tissue constructs.
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http://dx.doi.org/10.1177/2041731419859157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651657PMC
July 2019

Pilus-1 Backbone Protein RrgB of Streptococcus pneumoniae Binds Collagen I in a Force-Dependent Way.

ACS Nano 2019 06 11;13(6):7155-7165. Epub 2019 Jun 11.

Center for Applied Tissue Engineering and Regenerative Medicine , Munich University of Applied Sciences , 80335 Munich , Germany.

Attachment to host tissue is a prerequisite for successful host colonization and invasion of pathogens. Many pathogenic bacteria use surface appendices, called pili, to bind and firmly attach to host tissue surfaces. Although it has been speculated that the laterally positioned D3 domain of the pilus-1 backbone protein RrgB of Streptococcus pneumoniae may promote bacterial-host interaction, via adhesion to extracellular matrix molecules, such as collagen, earlier studies showed no affinity of RrgB to collagen I. Using atomic force microscopy-based single molecule force spectroscopy combined with lateral force microscopy, we show that under mechanical load, RrgB in fact binds to human collagen I in a force-dependent manner. We observe exceptionally strong interactions, with interaction forces reaching as much as 1500 pN, and we show that high force loading and shearing rates enhance and further strengthen the interaction. In addition, the affinity of RrgB to collagen I under mechanical load not only depends on the orientation of the D3 domain but also on the orientation of the collagen fibrils, relative to the pulling direction. Both exceptionally high binding forces and force-induced bond strengthening resemble the behavior of so-called catch bonds, which have recently been observed in bacterial adhesins, but have not been reported for multimeric backbone subunits of virulence related pili.
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http://dx.doi.org/10.1021/acsnano.9b02587DOI Listing
June 2019

Mechanical Activation Drastically Accelerates Amide Bond Hydrolysis, Matching Enzyme Activity.

Angew Chem Int Ed Engl 2019 07 24;58(29):9787-9790. Epub 2019 Jun 24.

Department of Applied Sciences and Mechatronics, Munich University of Applied Sciences, Lothstrasse 34, 80334, Munich, Germany.

Amide bonds, which include peptide bonds connecting amino acids in proteins and polypeptides, give proteins and synthetic polyamides their enormous strength. Although proteins and polyamides sustain mechanical force in nature and technology, how forces affect amide and peptide bond stability is still unknown. Using single-molecule force spectroscopy, we discover that forces of only a few hundred pN accelerate amide hydrolysis 10 -fold, an acceleration hitherto only known from proteolytic enzymes. The drastic acceleration at low force precedes a moderate additional acceleration at nN forces. Quantum mechanochemical ab initio calculations explain these experimental results mechanistically and kinetically. Our findings reveal that, in contrast to previous belief, amide stability is strongly force dependent. These calculations provide a fundamental understanding of the role of mechanical activation in amide hydrolysis and point the way to potential applications from the recycling of macromolecular waste to the design of bioengineered proteolytic enzymes.
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http://dx.doi.org/10.1002/anie.201902752DOI Listing
July 2019

Aggrecan Hypomorphism Compromises Articular Cartilage Biomechanical Properties and Is Associated with Increased Incidence of Spontaneous Osteoarthritis.

Int J Mol Sci 2019 Feb 26;20(5). Epub 2019 Feb 26.

Laboratory of Experimental Surgery and Regenerative Medicine, Clinic for General, Trauma and Reconstructive Surgery, Ludwig-Maximilians University, 80336 Munich, Germany.

The gene encoding the proteoglycan aggrecan () is abundantly expressed in cartilage during development and adulthood, and the loss or diminished deposition of the protein results in a wide range of skeletal malformations. Furthermore, aggrecan degradation is a hallmark of cartilage degeneration occurring in osteoarthritis. In the present study, we investigated the consequences of a partial loss of aggrecan in the postnatal skeleton and in the articular cartilage of adult mice. We took advantage of the previously described mouse line, which allows for conditional and timely-regulated deletion of floxed, cartilage-expressed genes. As previously reported, the introduction of the CreER cassette in the 3'UTR causes a disruption of the normal expression of resulting in a hypomorphic deposition of the protein. In homozygous mice, we observed a dwarf phenotype, which persisted throughout adulthood supporting the evidence that reduced aggrecan amount impairs skeletal growth. Homozygous mice exhibited reduced proteoglycan staining of the articular cartilage at 6 and 12 months of age, increased stiffening of the extracellular matrix at six months, and developed severe cartilage erosion by 12 months. The osteoarthritis in the hypomorph mice was not accompanied by increased expression of catabolic enzymes and matrix degradation neoepitopes. These findings suggest that the degeneration found in homozygous mice is likely due to the compromised mechanical properties of the cartilage tissue upon aggrecan reduction.
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http://dx.doi.org/10.3390/ijms20051008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429589PMC
February 2019

Age related changes in cell stiffness of tendon stem/progenitor cells and a rejuvenating effect of ROCK-inhibition.

Biochem Biophys Res Commun 2019 02 11;509(3):839-844. Epub 2019 Jan 11.

Center for Applied Tissue Engineering and Regenerative Medicine - CANTER, Munich University of Applied Sciences, Munich, Germany; Center for NanoScience, Ludwig-Maximilians-University, Munich, Germany. Electronic address:

Tendon stem/progenitor cells (TSPC) are potential targets for regenerative medicine and the treatment of tendon injuries. The frequency of such injuries increases in elderly patients while the proportion of functional TSPCs in tendon tissue decreases, protracting tendon repair. Using atomic force microscopy (AFM), we show that cell stiffness and size increase in TSPCs isolated from elderly patients (A-TSPC) compared to TSPCs from younger patients (Y-TSPC). Additionally, two-photon excited fluorescence (TPEF) microscopy revealed a denser, well-structured actin cytoskeleton in A-TSPC, which correlates with the augmented cell stiffness. Treating A-TSPC with ROCK-inhibitor, reverses these age-related changes, and has rejuvenating effect on cell morphology and stiffness. We assume that cellular stiffness is a suitable marker for cell aging and ROCK a potential target for therapeutic applications of cell rejuvenation.
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http://dx.doi.org/10.1016/j.bbrc.2019.01.027DOI Listing
February 2019

A Perfusion Bioreactor System for Cell Seeding and Oxygen-Controlled Cultivation of Three-Dimensional Cell Cultures.

Tissue Eng Part C Methods 2018 10;24(10):585-595

1 Center for Applied Tissue Engineering and Regenerative Medicine (CANTER), University of Applied Sciences Munich , Munich, Germany .

Bioreactor systems facilitate three-dimensional (3D) cell culture by coping with limitations of static cultivation techniques. To allow for the investigation of proper cultivation conditions and the reproducible generation of tissue-engineered grafts, a bioreactor system, which comprises the control of crucial cultivation parameters in independent-operating parallel bioreactors, is beneficial. Furthermore, the use of a bioreactor as an automated cell seeding tool enables even cell distributions on stable scaffolds. In this study, we developed a perfusion microbioreactor system, which enables the cultivation of 3D cell cultures in an oxygen-controlled environment in up to four independent-operating bioreactors. Therefore, perfusion microbioreactors were designed with the help of computer-aided design, and manufactured using the 3D printing technologies stereolithography and fused deposition modeling. A uniform flow distribution in the microbioreactor was shown using a computational fluid dynamics model. For oxygen measurements, microsensors were integrated in the bioreactors to measure the oxygen concentration (OC) in the geometric center of the 3D cell cultures. To control the OC in each bioreactor independently, an automated feedback loop was developed, which adjusts the perfusion velocity according to the oxygen sensor signal. Furthermore, an automated cell seeding protocol was implemented to facilitate the even distribution of cells within a stable scaffold in a reproducible way. As proof of concept, the human mesenchymal stem cell line SCP-1 was seeded on bovine cancellous bone matrix of 1 cm and cultivated in the developed microbioreactor system at different oxygen levels. The oxygen control was capable to maintain preset oxygen levels ±0.5% over a cultivation period of several days. Using the automated cell seeding procedure resulted in evenly distributed cells within a stable scaffold. In summary, the developed microbioreactor system enables the cultivation of 3D cell cultures in an automated and thus reproducible way by providing up to four independently operating, oxygen-controlled bioreactors. In combination with the automated cell seeding procedure, the bioreactor system opens up new possibilities to conduct more reproducible experiments to investigate optimal cultivation parameters and to generate tissue-engineering grafts in an oxygen-controlled environment.
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http://dx.doi.org/10.1089/ten.TEC.2018.0204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208160PMC
October 2018

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy.

J Vis Exp 2018 08 20(138). Epub 2018 Aug 20.

Center for Applied Tissue Engineering and Regenerative Medicine, Munich University of Applied Sciences; Center for Nano Science, Ludwig-Maximilians-Universität München.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.
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http://dx.doi.org/10.3791/58167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128213PMC
August 2018

Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses.

PLoS One 2018 2;13(5):e0195479. Epub 2018 May 2.

Lasercenter, Munich University of Applied Sciences, Lothstrasse, Munich, Germany.

Laser-induced cell transfer has been developed in recent years for the flexible and gentle printing of cells. Because of the high transfer rates and the superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of laser based cell printing. Therefore alternative approaches using deep UV laser sources and protein based acceptor films for energy absorption, have been introduced. Nevertheless, deep UV radiation can introduce DNA double strand breaks, thereby imposing the risk of carcinogenesis. Here we present a method for the laser-induced transfer of hydrogels and mammalian cells, which neither requires any sacrificial material for energy absorption, nor the use of UV lasers. Instead, we focus a near infrared femtosecond (fs) laser pulse (λ = 1030 nm, 450 fs) directly underneath a thin cell layer, suspended on top of a hydrogel reservoir, to induce a rapidly expanding cavitation bubble in the gel, which generates a jet of material, transferring cells and hydrogel from the gel/cell reservoir to an acceptor stage. By controlling laser pulse energy, well-defined cell-laden droplets can be transferred with high spatial resolution. The transferred human (SCP1) and murine (B16F1) cells show high survival rates, and good cell viability. Time laps microscopy reveals unaffected cell behavior including normal cell proliferation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195479PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931680PMC
July 2018

Single Molecule Force Spectroscopy Reveals Two-Domain Binding Mode of Pilus-1 Tip Protein RrgA of Streptococcus pneumoniae to Fibronectin.

ACS Nano 2018 01 9;12(1):549-558. Epub 2018 Jan 9.

Center for NanoScience, Ludwig-Maximilians-Universität München , 80799 Munich, Germany.

For host cell adhesion and invasion, surface piliation procures benefits for bacteria. A detailed investigation of how pili adhere to host cells is therefore a key aspect in understanding their role during infection. Streptococcus pneumoniae TIGR 4, a clinical relevant serotype 4 strain, is capable of expressing pilus-1 with terminal RrgA, an adhesin interacting with host extracellular matrix (ECM) proteins. We used single molecule force spectroscopy to investigate the binding of full-length RrgA and single RrgA domains to fibronectin. Our results show that full-length RrgA and its terminal domains D3 and D4 bind to fibronectin with forces of 51.6 (full length), 52.8 (D3), and 46.2 pN (D4) at force-loading rates of around 1500 pN/s. Selective saturation of D3 and D4 binding sites on fibronectin showed that both domains can interact simultaneously with fibronectin, revealing a two-domain binding mechanism for the pilus-1 tip protein. The high off rates and the corresponding short lifetime of the RrgA Fn bond (τ = 0.26 s) may enable piliated pneumococci to form and maintain a transient contact to fibronectin-containing host surfaces and thus to efficiently scan the surface for specific receptors promoting host cell adhesion and invasion. These molecular properties could be essential for S. pneumoniae pili to mediate initial contact to the host cells and-shared with other piliated Gram-positive bacteria-favor host invasion.
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http://dx.doi.org/10.1021/acsnano.7b07247DOI Listing
January 2018

Tenomodulin is Required for Tendon Endurance Running and Collagen I Fibril Adaptation to Mechanical Load.

EBioMedicine 2017 Jun 5;20:240-254. Epub 2017 May 5.

Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University (LMU), 80336 Munich, Germany; Experimental Trauma Surgery, Department of Trauma Surgery, University Regensburg Medical Centre, 93053 Regensburg, Germany. Electronic address:

Tendons are dense connective tissues that attach muscles to bone with an indispensable role in locomotion because of their intrinsic properties of storing and releasing muscle- generated elastic energy. Tenomodulin (Tnmd) is a well-accepted gene marker for the mature tendon/ligament lineage and its loss-of -function in mice leads to a phenotype with distinct signs of premature aging on tissue and stem/progenitor cell levels. Based on these findings, we hypothesized that Tnmd might be an important factor in the functional performance of tendons. Firstly, we revealed that Tnmd is a mechanosensitive gene and that the C-terminus of the protein co-localize with collagen I-type fibers in the extracellular matrix. Secondly, using an endurance training protocol, we compared Tnmd knockout mice with wild types and showed that Tnmd deficiency leads to significantly inferior running performance that further worsens with training. In these mice, endurance running was hindered due to abnormal response of collagen I cross-linking and proteoglycan genes leading to an inadequate collagen I fiber thickness and elasticity. In sum, our study demonstrates that Tnmd is required for proper tendon tissue adaptation to endurance running and aids in better understanding of the structural-functional relationships of tendon tissues.
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http://dx.doi.org/10.1016/j.ebiom.2017.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478207PMC
June 2017

Structural decoding of netrin-4 reveals a regulatory function towards mature basement membranes.

Nat Commun 2016 11 30;7:13515. Epub 2016 Nov 30.

Institute for Dental Research and Oral Musculoskeletal Biology, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, Cologne 50931, Germany.

Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.
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http://dx.doi.org/10.1038/ncomms13515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141367PMC
November 2016

Mechanochemical Cycloreversion of Cyclobutane Observed at the Single Molecule Level.

Chemistry 2016 Aug 14;22(34):12034-9. Epub 2016 Jul 14.

Institut für Ionenphysik und Angewandte Physik, Leopold-Franzens-Universität Innsbruck, Technikerstraße 25, 6020, Innsbruck, Austria.

Mechanochemical cycloreversion of cyclobutane is known from ultrasound experiments. It is, however, not clear which forces are required to induce the cycloreversion. In atomic force microscopy (AFM) experiments, on the other hand, it is notoriously difficult to assign the ruptured bond. We have solved this problem through the synthesis of tailored macrocycles, in which the cyclobutane mechanophore is bypassed by an ethylene glycol chain of specific length. This macrocycle is covalently anchored between a glass substrate and an AFM cantilever by polyethylene glycol linkers. Upon mechanical stretching of the macrocycle, cycloreversion occurs, which is identified by a defined length increase of the stretched polymer. The measured length change agrees with the value calculated with the external force explicitly included (EFEI) method. By using two different lengths for the ethylene glycol safety line, the assignment becomes unambiguous. Mechanochemical cycloreversion of cyclobutane is observed at forces above 1.7 nN.
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http://dx.doi.org/10.1002/chem.201600866DOI Listing
August 2016

Forced exercise-induced osteoarthritis is attenuated in mice lacking the small leucine-rich proteoglycan decorin.

Ann Rheum Dis 2017 Feb 4;76(2):442-449. Epub 2016 Jul 4.

Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany.

Objective: Interterritorial regions of articular cartilage matrix are rich in decorin, a small leucine-rich proteoglycan and important structural protein, also involved in many signalling events. Decorin sequesters transforming growth factor β (TGFβ), thereby regulating its activity. Here, we analysed whether increased bioavailability of TGFβ in decorin-deficient (Dcn) cartilage leads to changes in biomechanical properties and resistance to osteoarthritis (OA).

Methods: Unchallenged knee cartilage was analysed by atomic force microscopy (AFM) and immunohistochemistry. Active transforming growth factor β-1 (TGFβ1) content within cultured chondrocyte supernatants was measured by ELISA. Quantitative real-time (RT)-PCR was used to analyse mRNA expression of glycosaminoglycan (GAG)-modifying enzymes in C28/I2 cells following TGFβ1 treatment. In addition, OA was induced in Dcn and wild-type (WT) mice via forced exercise on a treadmill.

Results: AFM analysis revealed a strikingly higher compressive stiffness in Dcn than in WT cartilage. This was accompanied by increased negative charge and enhanced sulfation of GAG chains, but not by alterations in the levels of collagens or proteoglycan core proteins. In addition, decorin-deficient chondrocytes were shown to release more active TGFβ1. Increased TGFβ signalling led to enhanced Chst11 sulfotransferase expression inducing an increased negative charge density of cartilage matrix. These negative charges might attract more water resulting in augmented compressive stiffness of the tissue. Therefore, decorin-deficient mice developed significantly less OA after forced exercise than WT mice.

Conclusions: Our study demonstrates that the disruption of decorin-restricted TGFβ signalling leads to higher stiffness of articular cartilage matrix, rendering joints more resistant to OA. Therefore, the loss of an important structural component can improve cartilage homeostasis.
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http://dx.doi.org/10.1136/annrheumdis-2016-209319DOI Listing
February 2017

Structural and mechanical properties of the proliferative zone of the developing murine growth plate cartilage assessed by atomic force microscopy.

Matrix Biol 2016 Mar 8;50:1-15. Epub 2015 Oct 8.

Center for Applied Tissue Engineering and Regenerative Medicine (CANTER), Munich University of Applied Sciences, Munich, Germany; Center for NanoScience (CeNS), University of Munich, Munich, Germany. Electronic address:

The growth plate (GP) is a dynamic tissue driving bone elongation through chondrocyte proliferation, hypertrophy and matrix production. The extracellular matrix (ECM) is the major determinant of GP biomechanical properties and assumed to play a pivotal role for chondrocyte geometry and arrangement, thereby guiding proper growth plate morphogenesis and bone elongation. To elucidate the relationship between morphology and biomechanics during cartilage morphogenesis, we have investigated age-dependent structural and elastic properties of the proliferative zone of the murine GP by atomic force microscopy (AFM) from the embryonic stage to adulthood. We observed a progressive cell flattening and arrangement into columns from embryonic day 13.5 until postnatal week 2, correlating with an increasing collagen density and ECM stiffness, followed by a nearly constant cell shape, collagen density and ECM stiffness from week 2 to 4 months. At all ages, we found marked differences in the density and organization of the collagen network between the intracolumnar matrix, and the intercolumnar matrix, associated with a roughly two-fold higher stiffness of the intracolumnar matrix compared to the intercolumnar matrix. This difference in local ECM stiffness may force the cells to arrange in a columnar structure upon cell division and drive bone elongation during embryonic and juvenile development.
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http://dx.doi.org/10.1016/j.matbio.2015.10.001DOI Listing
March 2016

Decoding Cytoskeleton-Anchored and Non-Anchored Receptors from Single-Cell Adhesion Force Data.

Biophys J 2015 Oct;109(7):1330-3

Center for NanoScience, Ludwig-Maximilians-Universität München, Munich, Germany; Center for Applied Tissue Engineering and Regenerative Medicine, University of Applied Sciences, Munich, Germany. Electronic address:

Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking β1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that β1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions.
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http://dx.doi.org/10.1016/j.bpj.2015.07.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601042PMC
October 2015
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