RESUMO
Currently available heart valve replacements are limited in long-term performance or fail due to leaflet thickening, lack of growth or remodeling potential. In order to address these issues, it is necessary to mimic multiple factors of the native valvular extracellular matrix (ECM) such as architecture, mechanical behavior and biochemical signals. Here, we successfully generated an electrospun PEGdma-PLA scaffold adapted to the structure and mechanical properties of native valve leaflets. Valvular interstitial cells (VICs) and valvular endothelial cells (VECs) were seeded on the scaffold and when cultured under physiological conditions in a bioreactor, the construct performed like a native leaflet. Atomic force microscopy (AFM) was employed to obtain detailed mechanical information from the leaflets, which enabled the first layer-specific measurement of the Young's modulus. Interestingly, spongiosa stiffness was much lower compared to the fibrosa and ventricularis. Moreover, investigations into human fetal heart valve development identified collagen type I and versican as important structural proteins. As a proof of principle, these proteins were introduced to the scaffold, demonstrating the ability to bio-functionalize the hybrid valve based on natures' blueprint.
Assuntos
Próteses Valvulares Cardíacas , Engenharia Tecidual , Animais , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Suínos , Alicerces TeciduaisRESUMO
Semilunar valve leaflets have a well-described trilaminar histoarchitecture, with a sophisticated elastic fiber network. It was previously proposed that elastin-containing fibers play a subordinate role in early human cardiac valve development; however, this assumption was based on data obtained from mouse models and human second and third trimester tissues. Here, we systematically analyzed tissues from human fetal first (4-12 weeks) and second (13-18 weeks) trimester, adolescent (14-19 years) and adult (50-55 years) hearts to monitor the temporal and spatial distribution of elastic fibers, focusing on semilunar valves. Global expression analyses revealed that the transcription of genes essential for elastic fiber formation starts early within the first trimester. These data were confirmed by quantitative PCR and immunohistochemistry employing antibodies that recognize fibronectin, fibrillin 1, 2 and 3, EMILIN1 and fibulin 4 and 5, which were all expressed at the onset of cardiac cushion formation (~week 4 of development). Tropoelastin/elastin protein expression was first detectable in leaflets of 7-week hearts. We revealed that immature elastic fibers are organized in early human cardiovascular development and that mature elastin-containing fibers first evolve in semilunar valves when blood pressure and heartbeat accelerate. Our findings provide a conceptual framework with the potential to offer novel insights into human cardiac valve development and disease.
Assuntos
Elastina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Valvas Cardíacas/embriologia , Adolescente , Elasticidade , Elastina/biossíntese , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da Gravidez , Fatores de Tempo , Tropoelastina/biossíntese , Tropoelastina/metabolismo , Adulto JovemRESUMO
Investigations involving rare human tissues that are difficult to acquire due to their scarcity are highly challenging. The need to verify microarray analysis data by additional methods such as immunohistochemical staining and quantitative PCR creates an even greater demand for these valuable tissues. Furthermore, since rare human tissues may come from different sources and may have been processed by variable methods, the comparability of these samples must be verified. The aim of this study was to determine and validate a processing method that allows the analysis of human fetal and adult cardiovascular tissues from different sources that were preserved using varying methods. Due to restricted access to fresh human tissues and the need to accumulate these samples over an extended period of time, we used formalin-fixed paraffin-embedded tissues for gene expression analyses. We analyzed RNA levels from four different age groups: fetal first and second trimester, adolescents, and adults. In this study, we present an improved standard processing procedure for tissue sample processing and analysis of rare human cardiovascular tissues.
Assuntos
Feto/metabolismo , Perfilação da Expressão Gênica , RNA/isolamento & purificação , Biologia Computacional , Formaldeído , Humanos , Análise em Microsséries , RNA/análise , Fixação de TecidosRESUMO
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.
Assuntos
Técnicas Citológicas/métodos , Matriz Extracelular/química , Análise Espectral Raman/métodos , Animais , Proteínas da Matriz Extracelular/química , SuínosRESUMO
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.
Assuntos
Valva Aórtica/patologia , Colágeno Tipo I/química , Matriz Extracelular/metabolismo , Imunofluorescência/métodos , Análise Espectral Raman/métodos , Animais , Valva Aórtica/anormalidades , Valva Aórtica/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/ultraestrutura , Colagenases/metabolismo , Criopreservação/métodos , Matriz Extracelular/patologia , Matriz Extracelular/ultraestrutura , SuínosRESUMO
Our focus was to develop a three-dimensional (3D) human dynamic in vitro tissue model that mimics the natural microenvironment of the small intestine. We co-cultured human Caco-2 cells with primary-isolated human microvascular endothelial cells (hMECs) on decellularized porcine jejunal segments within a custom-made dynamic bioreactor system that resembles the apical and basolateral side of the intestine for up to 14 days. The obtained data were compared to results generated using routine static Caco-2 assays. We performed histology and immunohistochemistry. Permeability was measured using directed transport studies. Histological analyses revealed that in tissue-engineered segments, which had been cultured under dynamic conditions, the Caco-2 cells showed a high-prismatic morphology, resembling normal primary enterocytes within their native environment. We further identified that the transport of low permeable substances, such as fluorescein and desmopressin increased within the dynamic bioreactor cultures. Immunohistochemical staining showed a significantly higher expression of the efflux transport p-glycoprotein (p-gp) under dynamic culture conditions when compared to the static cultures. We conclude that the integration of physiological parameters is crucial for the establishment of a reliable 3D intestinal in vitro model, which enables the simulation of drug transport over the gut-blood-barrier in a simplified way.
Assuntos
Absorção Intestinal , Intestino Delgado/metabolismo , Modelos Biológicos , Reatores Biológicos , Células CACO-2 , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , HumanosRESUMO
Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues, their role in the cardiovascular system is poorly understood. In this study, we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and ß-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures, which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent ß-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that can serve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology.
Assuntos
Sistema Cardiovascular/embriologia , Células-Tronco Embrionárias/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Coração/embriologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Microscopia Confocal , Gravidez , beta Catenina/metabolismoRESUMO
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.
Assuntos
Materiais Biocompatíveis/química , Linhagem da Célula/fisiologia , Matriz Extracelular/fisiologia , Nicho de Células-Tronco/citologia , Nicho de Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Animais , HumanosRESUMO
Electrospinning is a long-known polymer processing technique that has received more interest and attention in recent years due to its versatility and potential use in the field of biomedical research. The fabrication of three-dimensional (3D) electrospun matrices for drug delivery and tissue engineering is of particular interest. In the present study, we identified optimal conditions to generate novel electrospun polymeric scaffolds composed of poly-D/L-lactide and poly-L-lactide in the ratio 50:50. Scanning electron microscopic analyses revealed that the generated poly(D/L-lactide-co-L-lactide) electrospun hybrid microfibers possessed a unique porous high surface area mimicking native extracellular matrix (ECM). To assess cytocompatibility, we isolated dermal fibroblasts from human skin biopsies. After 5 days of in vitro culture, the fibroblasts adhered, migrated and proliferated on the newly created 3D scaffolds. Our data demonstrate the applicability of electrospun poly(D/L-lactide-co-L-lactide) scaffolds to serve as substrates for regenerative medicine applications with special focus on skin tissue engineering.