The elucidation of biomechanics furthers our understanding of brain tumour biology. Brillouin spectroscopy is a new optical method that addresses viscoelastic properties down to subcellular resolution in a contact-free manner. Moreover, it can be combined with Raman spectroscopy to obtain co-localized biochemical information. Here, we applied co-registered Brillouin and Raman spectroscopy to U87-MG human glioblastoma cells in vitro. Using two-dimensional and three-dimensional cultures, we related biomechanical properties to local biochemical composition at the subcellular level, as well as the cell phenotype. Brillouin and Raman mapping of adherent cells showed that the nucleus and nucleoli are stiffer than the perinuclear region and the cytoplasm. The biomechanics of the cell cytoplasm is affected by culturing conditions, i.e. cells grown as spheroids are stiffer than adherent cells. Inside the spheroids, the presence of lipid droplets as assessed by Raman spectroscopy revealed higher Brillouin shifts that are not related to a local increase in stiffness, but are due to a higher refractive index combined with a lower mass density. This highlights the importance of locally defined biochemical reference data for a correct interpretation of the Brillouin shift of cells and tissues in future studies investigating the biomechanics of brain tumour models by Brillouin spectroscopy.

BACKGROUND: Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. OBJECTIVE: BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. METHODS: Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young’s modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). RESULTS: SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young’s modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. CONCLUSION: BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Glioblastoma (GBM) is the most aggressive form of malignant gliomas, with the eicosanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) playing a pivotal role in its progression via the COX-2/prostaglandin E2/4 axis. COX-2 upregulations in tumor cells induces a pro-inflammatory tumor microenvironment (TME), affecting the behavior of invading bone marrow-derived macrophages (Mϕ) and brain-resident microglia (MG) through unclear autocrine and paracrine mechanisms. Using CRISPR/Cas9 technology, we generated COX-2 knockout U87 glioblastoma cells. In spheroids and in vivo xenografts, this resulted in a significant inhibition of tumorigenic properties, while not observed in standard adherent monolayer culture. Here, the knockout induced a G1 cell cycle arrest in adherent cells, accompanied by increased ROS, mitochondrial activity, and cytochrome c-mediated apoptosis. In spheroids and xenograft models, COX-2 knockout led to notable growth delays and increased cell death, characterized by features of both apoptosis and autophagy. Interestingly, these effects were partially reversed in subcutaneous xenografts after co-culture with Mϕ, while co-culture with MG enhanced the growth-suppressive effects. In an orthotopic model, COX-2 knockout tumors displayed reduced proliferation (fewer Ki-67 positive cells), increased numbers of GFAP-positive astrocytes, and signs of membrane blebbing. These findings highlight the potential of COX-2 knockout and suppression as a therapeutic strategy in GBM, particularly when combined with suppression of infiltrating macrophages and stabilization of resident microglia populations to enhance anti-tumor effects.

Glutaraldehyde (GA)-treated bovine pericardium is still the gold standard for the fabrication of bioprostheses needed for the surgical treatment of valvular malfunction. Although excellent stability and low immunogenicity are accomplished, the application of GA is considered to be causal for structural valve deterioration, diminishing the long-term durability of bioprosthetic tissue. The novel GA-free SULEEI-treatment of bovine pericardium combines decellularization, riboflavin/UVA-cross-linking, and low-energy electron beam irradiation. In the present study, we initiated an in vivo application. We used a subcutaneous rat model to compare the immune and tissue responses, calcification propensity, and biomechanical properties of the alternatively prepared SULEEI bovine pericardial tissue with standard glutaraldehyde-fixed and industrially produced bovine pericardial patch material. SULEEI pericardium evokes a similar immune reaction and tissue response as the control standard bovine patch material. The calcification propensity of SULEEI tissue was low, and biomechanical analysis revealed a heterogeneous but similar pattern in tissue stiffness compared to the control patch. The results of this study highlight the potential of SULEEI-treated bovine pericardial tissue as a candidate for cutting-edge cardiovascular and valvular biomaterials in reconstructive surgery.

Recently, Brillouin spectroscopy has been proposed as a promising non-invasive tool to evaluate corneal biomechanics, e.g., during corneal cross-linking (CXL) treatment. However, the impact of corneal hydration on the Brillouin shift hampers straightforward interpretation of the measurements, especially when judging on the success of the CXL procedure. Therefore, in this work, we first quantify the effect of corneal (de)hydration on the Brillouin shift revealing that reliable measurements are only possible under constant hydration conditions, which was subsequently achieved by immersing porcine eyes in solution and waiting until an equilibrium state was reached. Investigations showed that Brillouin spectroscopy evaluates the CXL effect mainly indirectly via reduced water uptake, while polarization-sensitive optical coherence tomography evaluates the CXL effect directly via changes in collagen fiber alignment and is therefore insensitive to corneal hydration. Raman spectroscopy is not indicating any alterations in the molecular structure revealing that new cross-links are not created due to the CXL procedure. Compared to large water retention in balanced salt solution, the missing water uptake in dextran-based (16%) solution hampers the evaluation of the CXL effect by Brillouin spectroscopy.

Investigations on porcine eyes by Brillouin-Raman spectroscopy and polarization-sensitive optical coherence tomography revealed that mechanical stiffening of corneal cross-linking therapy is based on fiber alignment and hydration, but not on changes in the molecular structure.