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Type 2 diabetes (T2D) constitutes a major public health problem, and despite prevention efforts, this pandemic disease is one of the deadliest diseases in the world. In 2022, 6.7 million patients with T2D died prematurely from vascular complications. Indeed, diabetes increases the risk of myocardial infarction or stroke eightfold. The identification of the molecular factors involved in the occurrence of cardiovascular complications and their prevention are therefore major axes. Our hypothesis is that factors brought into play during physiological aging appear prematurely with diabetes progression. Our study focused on the aging of the extracellular matrix (ECM), a major element in the maintenance of vascular homeostasis. We characterized the morphological and functional aspects of aorta, with a focus on the collagen and elastic fibers of diabetic mice aged from 6 mo to nondiabetic mice aged 6 mo and 20 mo. The comparison with the two nondiabetic models (young and old) highlighted an exacerbated activity of proteases, which could explain a disturbance in the collagen accumulation and an excessive degradation of elastic fibers. Moreover, the generation of circulating elastin-derived peptides reflects premature aging of the ECM. These extracellular elements contribute to the appearance of vascular rigidity, often the origin of pathologies such as hypertension and atherosclerosis. In conclusion, we show that diabetic mice aged 6 mo present the same characteristics of ECM wear as those observed in mice aged 20 mo. This accelerated aortic wall remodeling could then explain the early onset of cardiovascular diseases and, therefore, the premature death of patients with T2D. NEW & NOTEWORTHY Aortic elastic fibers of young (6-mo old) individuals with diabetes degrade prematurely and exhibit an appearance like that found in aged (20-mo old) nondiabetic mice. Exacerbated elastolysis and elastin-derived peptide production are characteristic elements, contributing to early aortic wall rigidity and hypertension development. Therefore, limiting this early aging could be a judicious therapeutic approach to reduce cardiovascular complications and premature death in patients with diabetes.

Fibrosis is a consequence of the pathological remodeling of extracellular matrix (ECM) structures in the connective tissue of an organ. It is often caused by chronic inflammation, which over time, progressively leads to an excess deposition of collagen type I (COL I) that replaces healthy tissue structures, in many cases leaving a stiff scar. Increasing fibrosis can lead to organ failure and death; therefore, developing methods that potentially allow real-time monitoring of early onset or progression of fibrosis are highly valuable. In this study, the ECM structures of diseased and healthy human tissue from multiple organs were investigated for the presence of fibrosis using routine histology and marker-independent Raman microspectroscopy and Raman imaging. Spectral deconvolution of COL I Raman spectra allowed the discrimination of fibrotic and non-fibrotic COL I fibers. Statistically significant differences were identified in the amide I region of the spectral subpeak at 1608 cm -1 , which was deemed to be representative for structural changes in COL I fibers in all examined fibrotic tissues. Raman spectroscopy-based methods in combination with this newly discovered spectroscopic biomarker potentially offer a diagnostic approach to non-invasively track and monitor the progression of fibrosis. STATEMENT OF SIGNIFICANCE: Current diagnosis of fibrosis still relies on histopathological examination with invasive biopsy procedures. Although, several non-invasive imaging techniques such as positron emission tomography, single-photon emission computed tomography and second harmonic generation are gradually employed in preclinical or clinical studies, these techniques are limited in spatial resolution and the morphological interpretation highly relies on individual experience and knowledge. In this study, we propose a non-destructive technique, Raman microspectroscopy, to discriminate fibrotic changes of collagen type I based on a molecular biomarker. The changes of the secondary structure of collagen type I can be identified by spectral deconvolution, which potentially can provide an automatic diagnosis for fibrotic tissues in the clinical applicaion., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence β-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of β-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on β-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting β-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves β-cell response as well as the expression of relevant BM proteins. Our data show that β-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support β-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation. Impact statement Expression analysis identifies hypoxia-induced pathological changes in extracellular matrix (ECM) homeostasis as potential targets to support β-cell transplants by encapsulation in biomaterials for the treatment of diabetes mellitus. A collagen-1 hydrogel is shown to attenuate the effect of hypoxia on β-cells and their ECM expression. The functionalization of the hydrogel with endothelial cells increases the β-cell response to glucose and rescues essential basement membrane proteins.

The cells and tissues of the human body are constantly exposed to exogenous and endogenous forces that are referred to as biomechanical cues. They guide and impact cellular processes and cell fate decisions on the nano-, micro- and macro-scale, and are therefore critical for normal tissue development and maintaining tissue homeostasis. Alterations in the extracellular matrix composition of a tissue combined with abnormal mechanosensing and mechanotransduction can aberrantly activate signaling pathways that promote disease development. Such processes are therefore highly relevant for disease modelling or when aiming for the development of novel therapies. In this mini review, we describe the main biomechanical cues that impact cellular fates. We highlight their role during development, homeostasis and in disease. We also discuss current techniques and tools that allow us to study the impact of biomechanical cues on cell and tissue development under physiological conditions, and we point out directions, in which in vitro biomechanics can be of use in the future., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cell-derived matrices (CDMs) provide an exogenous source of human extracellular matrix (ECM), with applications as cell delivery vehicles, substrate coatings for cell attachment and differentiation, and as biomaterial scaffolds. However, commercial application of CDMs has been hindered due to the prolonged culture time required for sufficient ECM accumulation. One approach to increasing matrix deposition in vitro is macromolecular crowding (MMC), which is a biophysical phenomenon that limits the diffusion of ECM precursor proteins, resulting in increased ECM accumulation at the cell layer. Hyaluronic acid (HA), a natural MMC highly expressed in vivo during fetal development, has been shown to play a role in ECM production, but has not been investigated as a macromolecule for increasing cell-mediated ECM deposition in vitro. In the current study, we hypothesized that HA can act as a MMC, and increase cell-mediated ECM production. Human dermal fibroblasts were cultured for 3, 7, or 14 days with 0%, 0.05%, or 0.5% high molecular weight HA. Ficoll 70/400 was used as a positive control. SDS-PAGE, Sircol, and hydroxyproline assays indicated that 0.05% HA-treated cultures had significantly higher mean collagen deposition at 14 days, whereas Ficoll 70/400-treated cultures had significantly lower collagen production compared to the HA and untreated controls. However, fluorescent immunostaining of ECM proteins and quantification of mean gray values did not indicate statistically significant differences in ECM production in HA or Ficoll 70/400-treated cultures compared to untreated controls. Raman imaging (a marker-free spectral imaging method) indicated that HA increased ECM deposition in human dermal fibroblasts. These results are consistent with decreases in CDM stiffness observed in Ficoll 70/400-treated cultures by atomic force microscopy. Overall, these results indicate that there are macromolecule- and cell type- dependent effects on matrix assembly, turnover, and stiffness in cell-derived matrices. STATEMENT OF SIGNIFICANCE: Cell-derived matrices (CDMs) are versatile biomaterials with many regenerative medicine applications, including as cell and drug delivery vehicles and scaffolds for wound healing and tissue regeneration. While CDMs have several advantages, their commercialization has been limited due to the prolonged culture time required to achieve CDM synthesis in vitro. In this study, we explored the use of hyaluronic acid (HA) as a macromolecular crowder in human fibroblast cell cultures to support production of CDM biomaterials. Successful application of macromolecular crowding will allow development of human cell-derived, xeno-free biomaterials that re-capitulate the native human tissue microenvironment., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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. In addition, Raman spectra and images of tissue-engineered SMC constructs were acquired. Routine techniques such as qPCR, histochemistry, histological and immunocytological staining were performed for comparative gene and protein expression analysis. We identified that SMCs of different tissue origins exhibited unique spectral information that allowed a separation of all groups of origin by multivariate data analysis (MVA). We were further able to non-invasively monitor phenotypic switching in cultured SMCs and assess the impact of different culture conditions on extracellular matrix remodeling in the tissue-engineered ring constructs. Interestingly, we identified that the Raman signature of the human SMC-based ring constructs was similar to the one obtained from native aortic tissue. We conclude that Raman microspectroscopic methods are promising tools to characterize cells and define cellular and extracellular matrix components on a molecular level. In this study, in situ measurements were marker-independent, fast, and identified cellular differences that were not detectable by established routine techniques. Perspectively, Raman microspectroscopy and MVA in combination with artificial intelligence can be suitable for automated quality monitoring of (stem) cell and cell-based tissue engineering products. STATEMENT OF SIGNIFICANCE: The accessibility of autologous blood vessels for surgery is limited. Tissue engineering (TE) aims to develop functional vascular replacements; however, no commercially available TE vascular graft (TEVG) exists to date. One limiting factor is the availability of a well-characterized and safe cell source. Smooth muscle cells (SMCs) are generally used for TEVGs. To engineer a TEVG, proliferating SMCs of the synthesizing phenotype are essential, whereas functional, sustainable TEVGs require SMCs of the contractile phenotype. SMC diversity and plasticity are therefore limiting factors, also for their quality monitoring and application in TE. In this study, Raman microspectroscopy and imaging combined with machine learning tools allowed the non-destructive, marker-independent characterization of SMCs, smooth muscle tissues and TE SMC-constructs. The spectral information was specific enough to distinguish for the first time the phenotypic switching in SMCs in real-time, and monitor the impact of culture conditions on ECM remodeling in the TE SMC-constructs., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In a variety of diseases, from benign to life-threatening ones, inflammation plays a major role. Monitoring the intensity and extent of a multifaceted inflammatory process has become a cornerstone in diagnostics and therapy monitoring. However, the current tools lack the ability to provide insight into one of its most crucial aspects, namely, the alteration of the extracellular matrix (ECM). Using a radiolabeled platelet glycoprotein VI-based ECM-targeting fusion protein (GPVI-Fc), we investigated how binding of GPVI-Fc on fibrous tissue could uncover the progression of several inflammatory disease models at different stages (rheumatoid arthritis, cutaneous delayed-type hypersensitivity, lung inflammation and experimental autoimmune encephalomyelitis). Methods: The fusion protein GPVI-Fc was covalently linked to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and subsequently labeled with 64 Cu. We analyzed noninvasively in vivo 64 Cu-GPVI-Fc accumulation in murine cutaneous delayed-type hypersensitivity, anti-glucose-6-phosphate isomerase serum-induced rheumatoid arthritis, lipopolysaccharide-induced lung inflammation and an experimental autoimmune encephalomyelitis model. Static and dynamic Positron Emission Tomography (PET) of the radiotracer distribution was performed in vivo , with ex vivo autoradiography confirmation, yielding quantitative accumulation and a distribution map of 64 Cu-GPVI-Fc. Ex vivo tissue histological staining was performed on harvested samples to highlight the fusion protein binding to collagen I, II and III, fibronectin and fibrinogen as well as the morphology of excised tissue. Results: 64 Cu-GPVI-Fc showed a several-fold increased uptake in inflamed tissue compared to control tissue, particularly in the RA model, with a peak 24 h after radiotracer injection of up to half the injected dose. Blocking and isotype control experiments indicated a target-driven accumulation of the radiotracer in the case of chronic inflammation. Histological analysis confirmed a prolonged accumulation at the inflammation site, with a pronounced colocalization with the different components of the ECM (collagen III and fibronectin notably). Binding of the fusion protein appeared to be specific to the ECM but unspecific to particular components. Conclusion: Imaging of 64 Cu-GPVI-Fc accumulation in the ECM matrix appears to be a promising candidate for monitoring chronic inflammation. By binding to exposed fibrous tissue (collagen, fibronectin, etc.) after extravasation, a new insight is provided into the fibrotic events resulting from a prolonged inflammatory state., Competing Interests: Competing interests: M. Gawaz is a shareholder of advanceCOR GmbH.

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. In this study, ECM structures of human colon carcinoma and normal (control) colon tissues were histologically identified. Using atomic force microscopy (AFM) nanoindentation, we show that the collagen-rich regions within the ECM of colon carcinoma tissues were significantly stiffer than the submucosal collagen-rich layer of control tissues. Screening of these regions with Raman microspectroscopy revealed significantly different molecular fingerprints for collagen fibers in colon carcinoma tissues compared to control tissues. We further showed an increased alignment of collagen fibers and elevated levels of GAG immuno-reactivity within the collagen network of colon carcinoma tissues. GAGs such as heparan sulfate and chondroitin sulfate were detected in significantly elevated levels in collagen fibers of carcinoma tissues. Moreover, immunodetection of the collagen-associated proteoglycan decorin was significantly decreased in carcinomas tissues of individual patients when compared with the corresponding control tissues. Overall a strong patient-to-patient variability was evident in the ECM composition, structure and biomechanics of individual colon carcinoma tissues. Although, biomechanical characteristics of tumor ECM were not directly impacted by GAG content, GAGs might play an important role during the mechanical and structural remodeling of pathological tumor ECM. To manipulate GAG expression and deposition in tumor microenvironments could represent a novel potential therapeutic strategy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Regenerative strategies such as stem cell-based therapies and tissue engineering applications are being developed with the aim to replace, remodel, regenerate or support damaged tissues and organs. In addition to careful cell type selection, the design of appropriate three-dimensional (3D) scaffolds is essential for the generation of bio-inspired replacement tissues. Such scaffolds are usually made of degradable or non-degradable biomaterials and can serve as cell or drug carriers. The development of more effective and efficient drug carrier systems is also highly relevant for novel cancer treatment strategies. In this review, we provide a summary of current approaches that employ ECM and ECM-like materials, or ECM-synthetic polymer hybrids, as biomaterials in the field of regenerative medicine. We further discuss the utilization of such materials for cell and drug delivery, and highlight strategies for their use as vehicles for cancer therapy., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Tissue-engineered organs and implants hold promise for the replacement of damaged and diseased organs. However, the foreign body response (FBR) is a major obstacle that compromises the function of tissue-engineered constructs, typically causing them to fail. Two components of FBR are an inflammatory response and a lack of vascularization. To overcome these limitations, a collagen system was developed to release interleukin-6 (IL-6) siRNA and endothelial nitric oxide synthase (eNOS) pDNA in a staggered manner. Hollow collagen microspheres were assembled into a collagen sphere-in-hydrogel system that displayed a staggered release profile in vitro. This system was assessed in vivo in a subcutaneous rat model. The doses of IL-6 siRNA and eNOS pDNA were first individually optimized for their ability to reduce the volume fraction of inflammatory cells (7 days) and increase the length density of blood vessels (14 days), respectively. The identified optimal doses were combined, and the ability of the system to decrease the volume fraction of inflammatory cells and increase the length density of blood vessels was confirmed at both 7 and 14 days. Analysis of the tissue using Raman microspectroscopy revealed that in addition to changes in inflammation and angiogenesis, there were also changes in the extracellular matrix (ECM) at seven days. While changes in sulfated glycosaminoglycan (sGAG) content of the ECM were not detected, changes in the binding of sGAG of the ECM to growth factors were observed. Two growth factors tested, VEGF165 and bFGF showed increased binding to sGAG extracted from eNOS pDNA-treated samples at seven days, increasing the angiogenic potential of the ECM. Thus, we observe that changes in the tissue in terms of the balance of inflammation and angiogenesis as well changes in the activity of sGAG of the ECM occurs following dual delivery of nucleic acids from the collagen sphere-in-hydrogel system., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.

Human in vitro-manufactured tissue and organ models can serve as powerful enabling tools for the exploration of fundamental questions regarding cell, matrix, and developmental biology in addition to the study of drug delivery dynamics and kinetics. To date, the development of a human model of the renal proximal tubule (PT) has been hindered by the lack of an appropriate cell source and scaffolds that allow epithelial monolayer formation and maintenance. Using extracellular matrices or matrix proteins, an in vivo-mimicking environment can be created that allows epithelial cells to exhibit their typical phenotype and functionality. Here, we describe an in vitro-engineered PT model. We isolated highly proliferative cells from cadaveric human kidneys (human kidney-derived cells [hKDCs]), which express markers that are associated with renal progenitor cells. Seeded on small intestinal submucosa (SIS), hKDCs formed a confluent monolayer and displayed the typical phenotype of PT epithelial cells. PT markers, including N-cadherin, were detected throughout the hKDC culture on the SIS, whereas markers of later tubule segments were weak (E-cadherin) or not (aquaporin-2) expressed. Basement membrane and microvilli formation demonstrated a strong polarization. We conclude that the combination of hKDCs and SIS is a suitable cell-scaffold composite to mimic the human PT in vitro.

Directing appropriate extracellular matrix remodeling is a key aim of regenerative medicine strategies. Thus, antifibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix remodeling following cutaneous injury. It was hypothesized that delivery of miR-29B from a collagen scaffold will efficiently modulate the extracellular matrix remodeling response and reduce maladaptive remodeling such as aggressive deposition of collagen type I after injury. The release of RNA from the scaffold was assessed and its ability to silence collagen type I and collagen type III expression was evaluated in vitro. When primary fibroblasts were cultured with scaffolds doped with miR-29B, reduced levels of collagen type I and collagen type III mRNA expression were observed for up to 2 weeks of culture. When the scaffolds were applied to full thickness wounds in vivo, reduced wound contraction, improved collagen type III/I ratios and a significantly higher matrix metalloproteinase (MMP)-8: tissue inhibitor of metalloproteinase (TIMP)-1 ratio were detected when the scaffolds were functionalized with miR-29B. Furthermore, these effects were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 versus 5 μg). This study shows a potential of combining exogenous miRs with collagen scaffolds to improve extracellular matrix remodeling following injury.

Raman spectroscopy is an established laser-based technology for the quality assurance of pharmaceutical products. Over the past few years, Raman spectroscopy has become a powerful diagnostic tool in the life sciences. Raman spectra allow assessment of the overall molecular constitution of biological samples, based on specific signals from proteins, nucleic acids, lipids, carbohydrates, and inorganic crystals. Measurements are non-invasive and do not require sample processing, making Raman spectroscopy a reliable and robust method with numerous applications in biomedicine. Moreover, Raman spectroscopy allows the highly sensitive discrimination of bacteria. Rama spectra retain information on continuous metabolic processes and kinetics such as lipid storage and recombinant protein production. Raman spectra are specific for each cell type and provide additional information on cell viability, differentiation status, and tumorigenicity. In tissues, Raman spectroscopy can detect major extracellular matrix components and their secondary structures. Furthermore, the non-invasive characterization of healthy and pathological tissues as well as quality control and process monitoring of in vitro-engineered matrix is possible. This review provides comprehensive insight to the current progress in expanding the applicability of Raman spectroscopy for the characterization of living cells and tissues, and serves as a good reference point for those starting in the field., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Decorin is a structural and functional proteoglycan (PG) residing in the complex network of extracellular matrix (ECM) proteins in many connective tissues. Depending on the protein core and the glycosaminoglycan chain, PGs support cell adhesion, migration, proliferation, differentiation, ECM assembly and growth factor binding. For applications in tissue engineering, it is crucial to develop reliable, ECM-mimicking biomaterials. Electrospinning is a suitable method for creating three-dimensional (3D), fibrillar scaffolds. While there are numerous reports on the electrospinning of proteins including collagen, to date, there are no reports on the electrospinning of PGs. In the following study, we used electrospinning to generate decorin-containing matrices for tracheal tissue engineering applications. The electrospun scaffolds were analyzed using scanning electron microscopy, atomic force microscopy, contact angle measurements and dynamic mechanical analysis. Additionally, we confirmed PG functionality with immunostaining and 1,9-dimethylmethylene blue. To determine cell-matrix-interactions, tracheal cells (hPAECs) were seeded and analyzed using an FOXJ1-antibody. Moreover, interactions of the electrospun scaffolds with immune-mediated mechanisms were analyzed in detail. To conclude, we demonstrated the feasibility of electrospinning of decorin to generate functional 3D scaffolds with low immunogenicity for hPAEC expansion. Our data suggest that these hybrid materials may be suitable as a substrate for tracheal tissue engineering., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.(1-5) However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.(6) As every chemical vibration is assigned to a specific Raman band (wavenumber in cm(-1)), each biological sample features a typical spectral pattern due to their inherent biochemical composition.(7-9) Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.(1) Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).(10) Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO(2)) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

The non-destructive and label-free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non-contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400-1800 cm(-1)), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof-of-principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically-degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non-destructive and non-contact tool to probe ECM state in situ., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Stem cells are defined as unspecialized cells that are capable of long term self-renewal and differentiation into specialized cell types. These unique properties make them an attractive cell source for regenerative medicine applications. Although the functions of various stem cells have been extensively studied in the development of organisms and in diseases, the specific factors and conditions that control stem cell fate, specifically the conditions that allow them to remain unspecialized, are not well studied. It has been suggested that adult stem cell survival and maintenance, as well as proliferation and differentiation, are controlled by the three-dimensional (3D) microenvironment, the so-called niche. Major functional niche components include supporting niche cells, growth-modulating soluble factors stored within the niches, and the extracellular matrix (ECM). In this article, we review work highlighting the growing complexity of stem cell-ECM interactions and their impact on the fields of biomaterials research and regenerative medicine.

We previously reported that lacrimal glands (LGs) of male non-obese diabetic (NOD) mice, an established mouse model of autoimmune inflammatory LG disease that displays many features of human LGs in patients afflicted with Sjögren's syndrome (SjS), exhibit significant degradation of extracellular matrix (ECM) structures as well as increased expression of matrix metalloproteinases (MMPs). The purpose of the current study was to expand the spectrum of proteases identified, to clarify their probable origin as well as to identify the contribution of these changes to disease pathogenesis. We explored in depth the changes in ECM structures and ECM protease expression at the onset of disease (6 weeks) versus late stage disease (18 weeks) in male NOD mouse LGs, relative to LGs of age-matched male NODscid, a severely immunocompromised congenic strain, and healthy BALB/c mice. LG tissues were examined using routine histological, immunohistochemical, Western Blot and gene expression analyses novel multiphoton imaging technologies. We further characterized the profile of infiltrating immune cells under each condition using flow cytometry. Our results show that the initial infiltrating cells at 6 weeks of age are responsible for increased MMP and cathepsin H expression and therefore initiate the LG ECM degradation in NOD mice. More importantly, NODscid mice exhibited normal LG ECM structures, indicating the lymphocytes seen in the LGs of NOD mice are responsible for the degradation of the LG ECM. The disease-related remodeling of LG ECM structures may play a crucial role in altering the acinar signaling environment, disrupting the signaling scaffolds within the cells, which are required to mobilize the exocytotic trafficking machinery, ultimately leading to a loss of LG function in patients afflicted with SjS., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Aims: To increase the supply, many countries harvest allograft valves from explanted hearts of transplant recipients with ischaemic (ICM) or dilated cardiomyopathy (DCM). This study determines the structural integrity of valves from cardiomyopathic hearts., Methods and Results: Extracellular matrix (ECM) was examined in human valves obtained from normal, ICM, and DCM hearts. To confirm if ECM changes were directly related to the cardiomyopathy, we developed a porcine model of chronic ICM. Histology and immunohistostaining, as well as non-invasive multiphoton and second harmonic generation (SHG) imaging revealed marked disruption of ECM structures in human valves from ICM and DCM hearts. The ECM was unaffected in valves from normal and acute ICM pigs, whereas chronic ICM specimens showed ECM alterations similar to those seen in ICM and DCM patients. Proteins and proteinases implicated in ECM remodelling, including Tenascin C, TGFbeta1, Cathepsin B, MMP2, were upregulated in human ICM and DCM, and porcine chronic ICM specimens., Conclusion: Valves from cardiomyopathic hearts showed significant ECM deterioration with a disrupted collagen and elastic fibre network. It will be important to determine the impact of this ECM damage on valve durability and calcification in vivo if allografts are to be used from these donors.

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(epsilon-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.

Multiphoton microscopy has become a powerful method for the artifact-free, nondestructive evaluation of deep-tissue cells and extracellular matrix (ECM) structures in their native environment. By interacting with highly non-centrosymmetric molecular assemblies such as fibrillar collagen, the non-linear process called second harmonic generation (SHG) has also proven to be an important diagnostic tool for the visualization of ECM compartments in situ with submicron resolution without the need for tissue processing. This review reports on applications of multiphoton-induced autofluorescence and SHG microscopy to identify collagen and elastic fiber orientation in native, tissue-engineered and processed, as well as healthy and diseased, tissues and organs. SHG signal profiling was used to quantify ECM damage in various cardiovascular and exocrine tissues, as well as cartilage. These novel imaging modalities open the general possibility of high-resolution in situ and more important in vivo imaging of ECM structures, cells and intracellular organelles in living intact tissues.

Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(epsilon-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.

Lacrimal glands (LGs) of male non-obese diabetic (NOD) mice display many features of human LGs in patients afflicted with the autoimmune disease Sjögren's syndrome (SS), including the loss of secretory functions and a lymphocytic infiltration into the glands by 4 months of age. So far, research has mainly focused on the intracellular events that are involved in initiating LG dysfunction; however, the impact of SS on extracellular matrix (ECM) structures of the diseased LGs has not yet been determined. In this study we identified and compared LG ECM formation and integrity of age-matched male healthy (BALB/c) and diseased (NOD) mice. LG tissues were examined using routine histological, biochemical, immunohistochemical and gene expression analysis. Multiphoton imaging and second-harmonic generation (SHG) microscopy permitted the non-invasive analysis of major LG ECM structures including collagen- and elastin-containing fibers. Biochemical testing demonstrated a significant loss of collagen, glycosaminoglycans and desmosine in NOD LGs when compared to healthy BALB/c LGs. Immunohistochemical staining and gene expression analysis confirmed this disease-related alteration of LG ECM structures. Furthermore, laser-induced autofluorescence and SHG microscopy revealed dramatic changes in the structural organization of most collagenous and elastic fibers of the diseased LG tissues that were more pronounced than those displayed by histological analysis. Our results clearly show an enhanced degradation of ECM proteins accompanied by the severe disorganization and deformation of ECM structures of diseased LG tissues. These new insights into the involvement of ECM degradation in SS may lead to novel therapies for patients suffering from dry eye disease.

The types of cell-matrix adhesions and the signals they transduce strongly affect the cell-phenotype. We hypothesized that cells sense and respond to the three-dimensionality of their environment, which could be modulated by nano-structures on silicon surfaces. Human foreskin fibroblasts were cultured on nano-structures with different patterns (nano-post and nano-grate) and heights for 3 days. The presence of integrin alpha(5), beta(1), beta(3), paxillin and phosphorylated FAK (pFAK) were detected by western blot and immunofluorescence. Integrin beta(3) exhibited stronger signals on nano-grates. pFAK and paxillin were observed as small dot-like patterns on the cell-periphery on nano-posts and as elongated and aligned patterns on nano-grates. Collectively, our observations highlighted the presence of focal (integrin beta(1), beta(3), pFAK, paxillin), fibrillar (integrin alpha(5), beta(1)) and 3-D matrix (integrin alpha(5), beta(1), paxillin) adhesions on nano-structures. The presented nano-structures offer interesting opportunities to study the interaction of cells with topographical features comparable to the size of extracellular matrix components.

Background: Cryopreservation of human tissues, particularly heart valves, is widespread in clinical practice although the effects of this process on underlying tissue structures and its potential impact on valve durability have been poorly studied. Multiphoton imaging and second-harmonic generation (SHG) microscopy permit high-resolution, noninvasive analysis of living tissues at a subcellular level. In the present study we used these novel imaging modalities to compare the effects of vitreous and frozen cryopreservation on the extracellular matrix (ECM) of cardiac tissues., Methods: Conventional histology, electron microscopy, and multiphoton imaging to obtain autofluorescence and SHG images were performed on cardiac tissues to characterize the ECM in fresh, vitrified, and frozen cryopreserved tissues., Results: Autofluorescence and particularly SHG images revealed that conventional frozen cryopreservation of cardiac valves, when compared with fresh or vitrified tissues, leads to the loss of normal ECM structures in valve leaflets. Similar results were found in all other cardiac tissues suggesting that structural deterioration of the ECM is a common consequence of frozen cryopreservation., Conclusions: Our results demonstrate that conventional cryopreservation, when compared with fresh or vitrified tissues, causes more destruction of normal ECM structure, which might contribute to eventual graft dysfunction. Whether vitrification preservation will translate into greater durability or less valve failure will need to be determined.

Background: Transplantation of cryopreserved allografts represents a well-established valve replacement option. Despite their clinical use for more than 40 years, the integrity of the extracellular matrix (ECM) of these valves after thawing has not been determined. The purpose of this study was to investigate and compare ECM structures of fresh and cryopreserved porcine heart valve leaflets with special emphasis on the condition of collagenous and elastic fibers., Methods: Pulmonary valves were excised from unprocessed porcine hearts under sterile conditions. After treatment with antibiotics, the valves were incubated in a cryoprotective solution, cryopreserved stepwise, and stored at -196 degrees C for 1 week. Two groups of heart valves (fresh untreated and thawed cryopreserved [each, n = 8]) were analyzed using biochemical (collagen, elastin, desmosine), histologic (hematoxylin-eosin, Movat-pentachrome, resorcin-fuchsin), and immunohistochemical (antibodies against collagen I, III, IV, and elastin) methods. Near-infrared femtosecond multiphoton laser scanning microscopy and second harmonic generation were used for high-resolution three-dimensional imaging of ECM structures., Results: Biochemical testing demonstrated similar amounts of collagen and desmosine, but a minor loss of elastin in the cryopreserved specimens. Conventional histology revealed almost comparable cell and ECM formations in fresh and cryopreserved valve leaflets. In contrast, laser-induced autofluorescence imaging showed substantial ultrastructural deterioration and disintegration of most collagenous structures. Second harmonic generation was not inducible., Conclusions: Conventional cryopreservation of heart valves is accompanied by serious alterations and destruction of leaflet ECM structures, specifically demonstrated by multiphoton imaging. Further in-depth studies to clarify the impact of alternative cryopreservation techniques proposed for clinical use, such as vitrification, are crucial.

Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.

Objective: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes. The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves. Methods: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries. Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days. Controls were grown over an equivalent time period under static conditions. Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin–eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength). Results: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix. Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions. Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls. Conclusion: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity. [Copyright &y& Elsevier]

The multidisciplinary research of tissue engineering utilizes biodegradable or decellularized scaffolds with autologous cell seeding. Objective of this study was to investigate the impact of different decellularization protocols on extracellular matrix integrity of xenogeneic tissue by means of multiphoton femtosecond laser scanning microscopy, biochemical and histological analysis. Pulmonary valves were dissected from porcine hearts and placed in a solution of trypsin–EDTA and incubated at 37 °C for either 5, 8, or 24 h, followed by a 24 h PBS washing. Native and decellularized valves were processed for histology, DNA, cell proliferation, matrix proteins and biomechanical testing. Multiphoton NIR laser microscopy has been applied for high-resolution 3D imaging of collagen and elastin. Distinct differences in several ECM components following decellularization time were observed. Total GAG contents decreased in a time-dependent manner, with o-sulfated GAGs being more susceptible to degradation than n-sulfated GAGs. Efficiency of insoluble collagen extraction increased proportionally with decellularization time, suggesting ECM-integrity may be compromised with prolonged incubation. Biomechanical testing revealed a gradual weakening of mechanical strength with increased decellularization time. The enzymatic decellularization process of heart valves revealed a time-dependent loss of cells, ECM components and biomechanical strength. In order to avoid any immune response a thorough decellularization of 24 h remains mandatory. [Copyright &y& Elsevier]