Non-Invasive Three-Dimensional Cell Analysis in Bioinks by Raman Imaging.

3D bioprinting is an emerging biofabrication strategy using bioinks, comprising cells and biocompatible materials, to produce functional tissue models. Despite progress in building increasingly complex objects, biological analyses in printed constructs remain challenging. Especially, methods that allow non-invasive and non-destructive evaluation of embedded cells are largely missing.

Marker-Independent Monitoring of and Degradation of Supramolecular Polymers Applied in Cardiovascular Tissue Engineering.

The equilibrium between scaffold degradation and neotissue formation, is highly essential for tissue engineering. Herein, biodegradable grafts function as temporal roadmap to guide regeneration. The ability to monitor and understand the dynamics of degradation and tissue deposition in cardiovascular graft materials is therefore of great value to accelerate the implementation of safe and sustainable tissue-engineered vascular grafts (TEVGs) as a substitute for conventional prosthetic grafts.

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials.

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing.

Imaging of α-Synuclein Aggregates in a Rat Model of Parkinson's Disease Using Raman Microspectroscopy.

A hallmark of Parkinson's disease (PD) is the formation of Lewy bodies in the brain. Lewy bodies are rich in the aggregated form of misfolded α-Synuclein (α-Syn). The brain from PD patients can only be analyzed after postmortem, therefore, limiting the diagnosis of PD to the manifestation of motor symptoms.

Distinct Effects of Heparin and Interleukin-4 Functionalization on Macrophage Polarization and In Situ Arterial Tissue Regeneration Using Resorbable Supramolecular Vascular Grafts in Rats.

Two of the greatest challenges for successful application of small-diameter in situ tissue-engineered vascular grafts are 1) preventing thrombus formation and 2) harnessing the inflammatory response to the graft to guide functional tissue regeneration. This study evaluates the in vivo performance of electrospun resorbable elastomeric vascular grafts, dual-functionalized with anti-thrombogenic heparin (hep) and anti-inflammatory interleukin 4 (IL-4) using a supramolecular approach. The regenerative capacity of IL-4/hep, hep-only, and bare grafts is investigated as interposition graft in the rat abdominal aorta, with follow-up at key timepoints in the healing cascade (1, 3, 7 days, and 3 months).

Raman microspectroscopy and Raman imaging reveal biomarkers specific for thoracic aortic aneurysms.

Aortic rupture and dissection are life-threatening complications of ascending thoracic aortic aneurysms (aTAAs), and risk assessment has been largely based on the monitoring of lumen size enlargement. Temporal changes in the extracellular matrix (ECM), which has a critical impact on aortic remodeling, are not routinely evaluated, and cardiovascular biomarkers do not exist to predict aTAA formation. Here, Raman microspectroscopy and Raman imaging are used to identify spectral biomarkers specific for aTAAs in mice and humans by multivariate data analysis (MVA).

Nidogen-1 Mitigates Ischemia and Promotes Tissue Survival and Regeneration.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet -cell transplantations, are often hindered by ischemic conditions.

Elastin-like hydrogel stimulates angiogenesis in a severe model of critical limb ischemia (CLI): An insight into the glyco-host response.

Critical limb ischemia (CLI) is characterized by the impairment of microcirculation, necrosis and inflammation of the muscular tissue. Although the role of glycans in mediating inflammation has been reported, changes in the glycosylation following muscle ischemia remains poorly understood. Here, a murine CLI model was used to show the increase of high mannose, α-(2, 6)-sialic acid and the decrease of hybrid and bisected N-glycans as glycosylation associated with the ischemic environment.

Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study.

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable.

Towards automation in biologics production via Raman micro-spectroscopy, laser-induced forward cell transfer and surface-enhanced Raman spectroscopy.

Mammalian cells have become the predominant expression system for the production of biopharmaceuticals due to their capabilities in posttranslational modifications. In recent years, the efficacy of these production processes has increased significantly through technical improvements. However, the state of the art in the development of producer cell lines includes many manual steps and is as such very time and cost consuming.

Donor age significantly influences the Raman spectroscopic biomolecular fingerprint of human pancreatic extracellular matrix proteins following collagenase-based digestion.

Despite the enormous advances in the field of clinical pancreatic islet transplantation over the past two decades, the human islet isolation procedure remains suboptimal. Islets are extracted (isolated) from the exocrine tissue of donor pancreases using neutral protease (NP) and collagenase-based enzymes, which digest the extracellular matrix (ECM) scaffold surrounding human islets. This process remains highly variable and current isolation enzyme blends are ineffective at digesting pancreases from younger donors with low body mass indexes (BMI).

Non-invasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model.

The increasing prevalence of diabetes, its heterogeneity, and the limited number of treatment options drive the need for physiologically relevant assay platforms with human genetic background that have the potential to improve mechanistic understanding and e\xpedite diabetes-related research and treatment. In this study, we developed an endocrine pancreas-on-a-chip model based on a tailored microfluidic platform, which enables self-guided trapping of single human pseudo-islets. Continuous, low-shear perfusion provides a physiologically relevant microenvironment especially important for modeling and monitoring of the endocrine function as well as sufficient supply with nutrients and oxygen.

Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation.

Non-invasive functional molecular phenotyping of human smooth muscle cells utilized in cardiovascular tissue engineering.

Smooth muscle cell (SMC) diversity and plasticity are limiting factors in their characterization and application in cardiovascular tissue engineering. This work aimed to evaluate the potential of Raman microspectroscopy and Raman imaging to distinguish SMCs of different tissue origins and phenotypes. Cultured human SMCs isolated from different vascular and non-vascular tissues as well as fixed human SMC-containing tissues were analyzed.

Marker-Independent In Situ Quantitative Assessment of Residual Cryoprotectants in Cardiac Tissues.

Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations.

Non-invasive characterization of hybrid gelatin:poly-l-lactide electrospun scaffolds using second harmonic generation and multiphoton imaging.

Hybrid scaffolds composed of synthetic polymers and naturally occurring components have become more relevant in the field of tissue engineering and regenerative medicine. Synthetic polymers are responsible for scaffold durability, strength and structural integrity; however, often do not provide biological signals. Introducing a biological component leads to more advanced and biocompatible scaffolds.

Improved long-term durability of allogeneic heart valves in the orthotopic sheep model.

Objectives: Frozen cryopreservation (FC) with the vapour phase of liquid nitrogen storage (-135°C) is a standard biobank technique to preserve allogeneic heart valves to enable a preferable allograft valve replacement in clinical settings. However, their long-term function is limited by immune responses, inflammation and structural degeneration. Ice-free cryopreserved (IFC) valves with warmer storage possibilities at -80°C showed better matrix preservation and decreased immunological response in preliminary short-term in vivo studies.

Biomechanical and biomolecular characterization of extracellular matrix structures in human colon carcinomas.

The extracellular matrix (ECM) is extensively remodeled in tumor tissues. Overproduction of collagens, pathological collagen crosslinking and alignment of fibers are major processes that ultimately result in an increased tissue stiffness. Although it is known that glycosaminoglycans (GAGs) play an important role in tumor signaling, their contribution to the biomechanical properties of tumor ECM is unknown.

Electroconductive Biohybrid Collagen/Pristine Graphene Composite Biomaterials with Enhanced Biological Activity.

Electroconductive substrates are emerging as promising functional materials for biomedical applications. Here, the development of biohybrids of collagen and pristine graphene that effectively harness both the biofunctionality of the protein component and the increased stiffness and enhanced electrical conductivity (matching native cardiac tissue) obtainable with pristine graphene is reported. As well as improving substrate physical properties, the addition of pristine graphene also enhances human cardiac fibroblast growth while simultaneously inhibiting bacterial attachment (Staphylococcus aureus).

Impact of T-cell-mediated immune response on xenogeneic heart valve transplantation: short-term success and mid-term failure.

Objectives: Allogeneic frozen cryopreserved heart valves (allografts or homografts) are commonly used in clinical practice. A major obstacle for their application is the limited availability in particular for paediatrics. Allogeneic large animal studies revealed that alternative ice-free cryopreservation (IFC) results in better matrix preservation and reduced immunogenicity.

Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals.

Cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.

Exogenous miR-29B Delivery Through a Hyaluronan-Based Injectable System Yields Functional Maintenance of the Infarcted Myocardium.

Myocardial infarction (MI) results in debilitating remodeling of the myocardial extracellular matrix (ECM). In this proof-of-principle study it was sought to modulate this aggressive remodeling by injecting a hyaluronic acid-based reservoir delivering exogenous microRNA-29B (miR-29B). This proof-of-principal study was executed whereby myocardial ischemia/reperfusion was performed on C57BL/6 mice for 45 min after which five 10 μL boluses of a hydrogel composed of thiolated hyaluronic acid cross-linked with poly (ethylene glycol) diacrylate, containing exogenous miR-29B as an active therapy, were injected into the border zone of the infarcted myocardium.

Raman Spectroscopic Analyses of Jaw Periosteal Cell Mineralization.

To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1 osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1 jaw periosteal cells (JPCs, 3 donors) was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy.

Raman microspectroscopy as a diagnostic tool for the non-invasive analysis of fibrillin-1 deficiency in the skin and in the in vitro skin models.

Unlabelled: Fibrillin microfibrils and elastic fibers are critical determinants of elastic tissues where they define as tissue-specific architectures vital mechanical properties such as pliability and elastic recoil. Fibrillin microfibrils also facilitate elastic fiber formation and support the association of epithelial cells with the interstitial matrix. Mutations in fibrillin-1 (FBN1) are causative for the Marfan syndrome, a congenital multisystem disorder characterized by progressive deterioration of the fibrillin microfibril/ elastic fiber architecture in the cardiovascular, musculoskeletal, ocular, and dermal system.