extracting-young-s-modulus-native-murine-pulmonary-basement-membranes

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy Derived Force Maps

Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of ...

Munich University of Applied Sciences

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.

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

Adipose-derived multipotent stem/progenitor cells (ASPCs) were shown to be ideal candidates for cell-based regenerative therapies. Yet, despite their huge potential, successful clinical applications are still rare. It was suggested that the efficacy of ASPCs at the recipient site depends on the vehicle of cell delivery. In this study, for preparation of a murine critical-size nerve defect model, we assessed the commercially available fibrin gel (ARTISS) as a potential cell carrier. In a thorough in vitro analysis, we investigated cell-fibrin interactions and analyzed the distribution and the long-term behavior of ASPCs cultivated in fibrin gel under normoxic and hypoxic conditions. ASPCs attached to the surface of a thin fibrin layer (two-dimensional condition) and spread with the abundant formation of actin stress fibers. Cells cultured within a fibrin matrix (three-dimensional condition) displayed a uniform distribution and formed interconnected networks while exhibiting strong cell-matrix interactions. Using time-lapse analysis, cells were found to migrate out of the gel and subsequently proliferated robustly both under hypoxic and normoxic conditions. During 14 days of culture in fibrin gel, ASPCs showed high viability, metabolic, and remodeling activities. At the end of the culture period, the fibrin matrix was degraded entirely accompanied by an upregulation of matrix metalloproteinases. In conclusion, fibrin gel stands out as a valuable biomaterial for delivering vital and active cells to damaged tissues. As a direct proof, ASPCs carried in a fibrin matrix will be evaluated in a murine critically sized peripheral nerve repair model.

Fibrin glue displays promising in vitro characteristics as a potential carrier of adipose progenitor cells for tissue regeneration.

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.

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

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.

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

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.

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

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.

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

Mammalian cells sense and react to the mechanics of their immediate microenvironment. Therefore, the characterization of the biomechanical properties of tissues with high spatial resolution provides valuable insights into a broad variety of developmental, homeostatic and pathological processes within living organisms. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) substructure measuring only ∼100-400 nm across, are, among other things, pivotal to tumor progression and metastasis formation. Although the precise assignment of the Young's modulus E of such a thin ECM substructure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young's modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryosectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young's modulus E using an in-house tailored user-friendly AFM data analysis software, the Center for Applied Tissue Engineering and Regenerative Medicine processing toolbox, which enables automatic loading of the recorded force maps, conversion of the force versus piezo-extension curves to force versus indentation curves, calculation of Young's moduli and generation of Young's modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1-2 d.

Article	Year
Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses. PloS one\| PubMed ID: 29718923	2018
Fibrin glue displays promising in vitro characteristics as a potential carrier of adipose progenitor cells for tissue regeneration. Journal of tissue engineering and regenerative medicine\| PubMed ID: 30515986	2019
Aggrecan Hypomorphism Compromises Articular Cartilage Biomechanical Properties and Is Associated with Increased Incidence of Spontaneous Osteoarthritis. International journal of molecular sciences\| PubMed ID: 30813547	2019
Pilus-1 Backbone Protein RrgB of Streptococcus pneumoniae Binds Collagen I in a Force-Dependent Way. ACS nano\| PubMed ID: 31184856	2019
Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics. Advanced healthcare materials\| PubMed ID: 33025765	2020
Early Detection of Cartilage Degeneration: A Comparison of Histology, Fiber Bragg Grating-Based Micro-Indentation, and Atomic Force Microscopy-Based Nano-Indentation. International journal of molecular sciences\| PubMed ID: 33036285	2020
Profiling native pulmonary basement membrane stiffness using atomic force microscopy. Nature protocols\| PubMed ID: 38429517	2024

Bastian Hartmann

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