Abstract Stem cells play a major role in human development, and postnatal tissues homeostasis, an... more Abstract Stem cells play a major role in human development, and postnatal tissues homeostasis, and repair. Therefore, they are considered as significant building blocks for tissue engineering and regenerative medicine applications. Stem cells receive important cues from their natural microenvironment, the niche, which determine each stem cell’s behavior and fate decision. The stem cell niche is composed of physico-chemical and cellular elements, which could originate from local or distant tissues, or from sources external to the body. In addition to the niche, the behavior of stem cells may be explained by their normal process of development. Therefore, an understanding of stem cells niche, the initial stages of development and sources and origins of stem cells is necessary to facilitate the design of protocols for the use of stem cells in tissue engineering and regenerative medicine. When these protocols involve in vitro manipulation of stem cells, the elements of niche components are usually used as the major players including soluble factors, extracellular matrix components, physical factors, and cells from other lineages. Also, the recently defined concepts of transdifferentiation and reprogramming have opened new possibilities for generation of further cell sources, especially from autologous tissues. There are, however, still many gaps in the knowledge of in vivo and in vitro stem cell biology, and in understanding the mechanisms of a number of diseases which will benefit from stem cell therapy. Filling these gaps will permit the progression from stem cells research to clinical applications.
Liver transplantation from compatible donors has been the main therapy available for patients wit... more Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.
Pulmonary regenerative medicine and tissue engineering is a rapidly expanding field combining the... more Pulmonary regenerative medicine and tissue engineering is a rapidly expanding field combining the core elements of stem cell biology, scaffold engineering, and design of multifaceted cell culture systems and bioreactors. Methods for derivation of lung epithelial cell lineages from embryonic stem cells have been established and are currently being refined; the existence of multipotent adult human lung stem cells that give rise to epithelial and vascular tissue has been reported. The absence of literature regarding derivation of lung-specific vascular and mesenchymal lineages presents a challenge moving forward. Methods of implementation and therapeutic efficacy of stem cell-derived distal pulmonary cell populations in animal models of human lung disease remain to be determined. The current ‘state of the art' for high-fidelity complex scaffolding in lung tissue engineering is the use of decellularized cadaveric lungs, which provides a unique platform for whole lung organ engineering. This approach allows for preservation of the intricate lung architecture that may be subsequently recellularized via perfusion through both the airway and vascular conduits. Implementation of engineered scaffolds from other sources will require methods that capture the unique architectural and mechanical properties of the lung. A major advance in the field has been the development of bioreactor systems capable of simultaneous airway ventilation and vascular perfusion of tissue-engineered lungs created using decellularized lung scaffolds. Scale-up of these systems to larger animal models and eventually humans, as well as the potential of lingering immunogenicity and availability of healthy cadaveric tissue remain major challenges facing this paradigm. Other key areas of research discussed in this chapter include the development of stem cell bioprocessing systems and microfluidic ‘lung-on-a-chip' devices that provide a novel platform for high throughput testing of pharmaceuticals.
In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secr... more In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secretion coupling, we have previously demonstrated that the actomyosin system might be involved in the transport of cations across the plasma membrane of bovine adrenal chromaffin cells [(1986) J. Biol. Chem. 261, 5745 57501. To establish whether actin and myosin might also be involved in later stages of the cellular response, we have examined the possible effects of various actin-specific reagents on the calcium-mediated secretion of catecholamines from digitonin-permeabilized cells. F-Actin-destabilizing agents, such as cytochalasin D or DNase 1, were found to promote Ca *+-stimulated (as well as basal) secretion. By contrast, stabilizers, like phalloidin, produced the opposite effect. It is concluded that stimulus-secretion coupling in chromaffin cells might require the reorganization of actin for modulating both ion transport across the plasma membrane and exocytotic secretion per se.
Despite the significant progress in engineering fairly thin tissues that contain or acquire vascu... more Despite the significant progress in engineering fairly thin tissues that contain or acquire vascular structures (eg skin, cartilage and bladder), it has been markedly difficult to construct metabolically demanding organs with thicker and more complex structures (eg heart, lung, kidney, and liver). This research explores a new generation of clinically significant (500cm) liver tissue constructs made of a new natural polymer composite, imbedded with optimized network of flow channels, and manufactured using innovative structured porogen and lost- ...
Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) condition... more Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.
Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as p... more Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as proliferation and migration of endothelial cells (ECs) from different vascular beds, induces capillary sprouting in chorioallantoic membrane and improves in vivo vascularization in a hind-limb ischemic model. In the present study, we sought to investigate the signaling properties of NGF in a microcapillary ECs model compared to those of a neuronal model. NGF-induced phosphorylation of signaling molecules Erk1/2, Akt and PLCγ were measured using Western blotting and compared between mouse NGF (mNGF) and snake venom NGF analogues. NGFs-induced signaling was TrkA mediated as evident by inhibition with the TrkA antagonist K252a. NGF and its analogues-induced signaling in ECs were characterized by a transient effect in contrast to a prolonged stimulation in neuronal cells. The potency of mouse, cobra and viper NGFs to induce Erk1/2 phosphorylation in ECs was higher than in neurons. In ECs, mNGF exhibited the highest efficacy of stimulation of Erk1/2 phosphorylation, followed by viper and cobra NGFs. The efficacy of stimulation of Erk1/ 2 phosphorylation measured with neurons was opposite from that in ECs. NGF-induced temporal signaling differences between ECs and neurons may explain the dual vascular and neurotrophic effects of this growth factor.
Biochimica Et Biophysica Acta - General Subjects, Jun 1, 2015
Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The... more Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The physiological role of this protein is not characterized. We report that a CRISP isolated from Echis carinatus sochureki venom (ES-CRISP) inhibits angiogenesis. The anti-angiogenic activity of purified ES-CRISP from snake venom was investigated in vitro using endothelial cells assays such as proliferation, migration and tube formation in Matrigel, as well as in vivo in quail embryonic CAM system. The modulatory effect of ES-CRISP on the expression of major angiogenesis factors and activation of angiogenesis pathways was tested by qRT-PCR and Western blot. The amino acid sequence of ES-CRISP was found highly similar to other members of this snake venom protein family, and shares over 50% identity with human CRISP-3. ES-CRISP supported adhesion to endothelial cells, although it was also internalized into the cytoplasm in a granule-like manner. It blocked EC proliferation, migration and tube formation in Matrigel. In the embryonic quail CAM system, ES-CRISP abolished neovascularization process induced by exogenous growth factors (bFGF, vpVEGF) and by developing gliomas. CRISP modulates the expression of several factors at the mRNA level, which were characterized as regulators of angiogenesis and blocked activation of MAPK Erk1/2 induced by VEGF. ES-CRISP was characterized as a negative regulator of the angiogenesis, by direct interaction with endothelial cells. The presented work may lead to the development of novel angiostatic therapy, as well as contribute to the identification of the physiological relevance of this functionally uncharacterized protein.
We investigated the effects of hypoxia on spontaneous (SP)-and activin A (AA)-induced definitive ... more We investigated the effects of hypoxia on spontaneous (SP)-and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O 2 , but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31-and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1a and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1a-/-mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1a-/-mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1a-/-mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.
Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoi... more Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and β1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ~5 μg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS.
Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) condition... more Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.
The intrinsic fluorescence of catecholamines can be exploited for a simple and speedy fluorescenc... more The intrinsic fluorescence of catecholamines can be exploited for a simple and speedy fluorescence assay of secretion from bovine adrenal chromaffin cells. Catecholamines are separated from concomitantly secreted proteins by centrifugation in 0.4 M perchloric acid, and their fluorescence is measured directly in a conventional spectrofluorometer without chemical derivatization. Catecholamine release, assayed by this technique, is virtually identical to data obtained in parallel by the classical trihydroxyindole method. Intrinsic catecholamine fluorescence does not differentiate between epinephrine and norepinephrine. Interferences from small molecular weight compounds not precipitated in perchloric acid are rare, and can be accounted for using appropriate calibration curves.
Anterior cruciate ligament (ACL) reconstructive surgery is a major health concern world-wide beca... more Anterior cruciate ligament (ACL) reconstructive surgery is a major health concern world-wide because of a large aging population and increased occurrence of sport-related damage. Tissue engineering is a rapidly growing interdisciplinary field that offers a promising new approach for ACL repair. In order to overcome the shortages of current existing surgical fixation devices, we are combining gradient cellular structure (GCS) injection molding technique and biomedical engineering to develop novel surgical fixation devices (screw, ...
Wolters Kluwer Health may email you for journal alerts and information, but is committed to maint... more Wolters Kluwer Health may email you for journal alerts and information, but is committed to maintaining your privacy and will not share your personal information without your express consent. For more information, please refer to our Privacy Policy. ... Skip Navigation Links Home > March/April 1997 - Volume 43 - Issue 2 > Exposing Endothelialized Cardiac Prosthesis to Pulsatile Flo... ... An abstract is unavailable. This article is available as a PDF only. ... Thought you might appreciate this item(s) I saw at ASAIO Journal. ... Your message has been successfully sent to your ...
Hemocompatibility of the blood-contacting surface of artificial cardiac prostheses remains a majo... more Hemocompatibility of the blood-contacting surface of artificial cardiac prostheses remains a major challenge for their long-term clinical use. We hypothesized that the thrombogenicity of segmented polyurethanes (PUS) used for creating the blood sacs can be reduced by lining the luminal surface with autologous endothelial cells (EC). Over the past years we have optimized our approach by 1. choosing an appropriate (detoxified) biomaterial (Biospan@), 2. generating a controlled, roughened PU surface and 3. precoating the luminal surface with an autologous protein complex (APC) made from plasma cryoprecipitate, 4. using autologous EC isolated from adipose tissue or the jugular vein, 5. designing a seeding device with 3-D rotation, which allows for the effective, uniform endothelialization of fully assembled cardiac prostheses,and 6. developing techniques for non-destructive monitoring of the extent of EC coverage. In vitro studies confirm the importance of exposing the EC lining the blood sacs to hemodynamic forces (flow and cyclic strain) prior to implantation for establishing a nonthrombogenic, shear-resistant EC monolayer. We recently tested fully endothelialized Ventricular Assist Devices (VADs) in vitro in a mock-loop circulation (3.2 l/min flow at 1 Hz) for 6 and 24 hours. At 6 hours, no denudation was observed. After 24 hours the integrity of the EC-monolayer on fibronectin-coated PUS was partially (<300/o) impaired. By contrast, virtually no denudation was observed when the cells were seeded on Biospan@ precoated with bovine cryoprecipitate. Similar results were obtained in our first er vivo tests in which fully endothelialized VADs were connected to an extracorporeal aorta-to aorta bypass in a calf model. Our results provide the first evidence for sucecssful in vitro and w vivo testing of endothelialized VADs and stress the importance of the composition of the extracellular matrix for maintaining a durable EC monolayer.
The Journal of Thoracic and Cardiovascular Surgery, 1995
Endothelial cell-lined in circulation skeletal muscle ventricles Skeletal muscle ventricles were ... more Endothelial cell-lined in circulation skeletal muscle ventricles Skeletal muscle ventricles were constructed from the latissimus dorsi in six dogs by wrapping the muscle around a polypropylene mandrel. Jugular vein endothelial cells were harvested enzymatically and grown in tissue culture. After 3 weeks of vascular delay and 4 weeks of electrical conditioning, five skeletal muscle ventricles were seeded with 5 to 8 x 106 autologous endothelial cells by percutaneous injection of a cellular suspension into the lumen of the skeletal muscle ventricle; one skeletal muscle ventricle was injected with culture medium alone as an unseeded control. The autologous endothelial cells were all prelabeled with a lipid-bound cellular marker, PKH-26. After an additional 4 weeks of electrical conditioning, the mandrels were removed and the skeletal muscle ventricles were connected to the descending thoracic aorta and activated to contract during cardiac diastole at a 1:2 ratio with the heart. After 3 hours of continuous pumping, mean diastolic pressure was increased by 35% (58-7 versus 78-6 mm Hg, p < 0.05). At this time, the skeletal muscle ventricles were excised for histologic examination. Sections stained with hematoxylin and eosin revealed a continuous cellular layer lining the skeletal muscle ventricle; no cells were present on the lumen of the control skeletal muscle ventricle. All seeded skeletal muscle ventricles exhibited fluorescence as a result of the PKH-26 cellular marker. Immunofluorescent staining with antibodies to von Willebrand factor and ultrastructural analysis with an electron microscope confirmed the endothelial character of these cells lining the lumen of the skeletal muscle ventricles. The ability to create endothelial cell-lined muscular pumping chambers holds important implications for the resolution of thrombotic events in cardiac assist devices as well as toward the clinical application of skeletal muscle ventricles. (J THORAC CARDIOVASC SURG 1995;109:66-73) Gregory A. Thomas, MD (by invitation), Peter I. Lelkes, PhD (by invitation), Susumu Isoda, MD, PhD (by invitation), Dawn Chick, BS (by invitation), Huiping Lu, MD (by invitation), Robert L. Hammond, BA (by invitation), Hidehiro Nakajima, MD, PhD (by invitation), Hisako Nakajima, MD (by invitation), Henry L. Waiters III, MD (by invitation), and
After nearly seven decades of development, dental composite restorations continue to show limited... more After nearly seven decades of development, dental composite restorations continue to show limited clinical service. The triggering point for restoration failure is the degradation of the bond at the tooth-biomaterial interface from chemical, biological, and mechanical sources. Oral biofilms form at the bonded interfaces, producing enzymes and acids that demineralize hard tissues and damage the composite. Removing bacteria from bonded interfaces and remineralizing marginal gaps will increase restorations' clinical service. To address this need, we propose for the first time the use of piezoelectric nanoparticles of barium titanate (BaTiO3) as a multifunctional bioactive filler in dental resin composites, offering combined antibacterial and (re)mineralization effects. In this work, we developed and characterized the properties of dental piezoelectric resin composites, including the degree of conversion and mechanical and physical properties, for restorative applications. Moreover, we evaluated the antibacterial and mineralization responses of piezoelectric composites in vitro. We observed a significant reduction in biofilm growth (up to 90%) and the formation of thick and dense layers of calcium phosphate minerals in piezoelectric composites compared to control groups. The antibacterial mechanism was also revealed. Additionally, we developed a unique approach evaluating the bond strength of dentin-adhesive-composite interfaces subjected to simultaneous attacks from bacteria and cyclic mechanical loading operating in synergy. Our innovative bioactive multifunctional composite provides an ideal technology for restorative applications using a single filler with combined long-lasting nonrechargeable antibacterial/remineralization effects.
Abstract Stem cells play a major role in human development, and postnatal tissues homeostasis, an... more Abstract Stem cells play a major role in human development, and postnatal tissues homeostasis, and repair. Therefore, they are considered as significant building blocks for tissue engineering and regenerative medicine applications. Stem cells receive important cues from their natural microenvironment, the niche, which determine each stem cell’s behavior and fate decision. The stem cell niche is composed of physico-chemical and cellular elements, which could originate from local or distant tissues, or from sources external to the body. In addition to the niche, the behavior of stem cells may be explained by their normal process of development. Therefore, an understanding of stem cells niche, the initial stages of development and sources and origins of stem cells is necessary to facilitate the design of protocols for the use of stem cells in tissue engineering and regenerative medicine. When these protocols involve in vitro manipulation of stem cells, the elements of niche components are usually used as the major players including soluble factors, extracellular matrix components, physical factors, and cells from other lineages. Also, the recently defined concepts of transdifferentiation and reprogramming have opened new possibilities for generation of further cell sources, especially from autologous tissues. There are, however, still many gaps in the knowledge of in vivo and in vitro stem cell biology, and in understanding the mechanisms of a number of diseases which will benefit from stem cell therapy. Filling these gaps will permit the progression from stem cells research to clinical applications.
Liver transplantation from compatible donors has been the main therapy available for patients wit... more Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5 weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.
Pulmonary regenerative medicine and tissue engineering is a rapidly expanding field combining the... more Pulmonary regenerative medicine and tissue engineering is a rapidly expanding field combining the core elements of stem cell biology, scaffold engineering, and design of multifaceted cell culture systems and bioreactors. Methods for derivation of lung epithelial cell lineages from embryonic stem cells have been established and are currently being refined; the existence of multipotent adult human lung stem cells that give rise to epithelial and vascular tissue has been reported. The absence of literature regarding derivation of lung-specific vascular and mesenchymal lineages presents a challenge moving forward. Methods of implementation and therapeutic efficacy of stem cell-derived distal pulmonary cell populations in animal models of human lung disease remain to be determined. The current ‘state of the art' for high-fidelity complex scaffolding in lung tissue engineering is the use of decellularized cadaveric lungs, which provides a unique platform for whole lung organ engineering. This approach allows for preservation of the intricate lung architecture that may be subsequently recellularized via perfusion through both the airway and vascular conduits. Implementation of engineered scaffolds from other sources will require methods that capture the unique architectural and mechanical properties of the lung. A major advance in the field has been the development of bioreactor systems capable of simultaneous airway ventilation and vascular perfusion of tissue-engineered lungs created using decellularized lung scaffolds. Scale-up of these systems to larger animal models and eventually humans, as well as the potential of lingering immunogenicity and availability of healthy cadaveric tissue remain major challenges facing this paradigm. Other key areas of research discussed in this chapter include the development of stem cell bioprocessing systems and microfluidic ‘lung-on-a-chip' devices that provide a novel platform for high throughput testing of pharmaceuticals.
In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secr... more In the search for a functional role of cytoskeletal proteins in the mechanism(s) of stimulus-secretion coupling, we have previously demonstrated that the actomyosin system might be involved in the transport of cations across the plasma membrane of bovine adrenal chromaffin cells [(1986) J. Biol. Chem. 261, 5745 57501. To establish whether actin and myosin might also be involved in later stages of the cellular response, we have examined the possible effects of various actin-specific reagents on the calcium-mediated secretion of catecholamines from digitonin-permeabilized cells. F-Actin-destabilizing agents, such as cytochalasin D or DNase 1, were found to promote Ca *+-stimulated (as well as basal) secretion. By contrast, stabilizers, like phalloidin, produced the opposite effect. It is concluded that stimulus-secretion coupling in chromaffin cells might require the reorganization of actin for modulating both ion transport across the plasma membrane and exocytotic secretion per se.
Despite the significant progress in engineering fairly thin tissues that contain or acquire vascu... more Despite the significant progress in engineering fairly thin tissues that contain or acquire vascular structures (eg skin, cartilage and bladder), it has been markedly difficult to construct metabolically demanding organs with thicker and more complex structures (eg heart, lung, kidney, and liver). This research explores a new generation of clinically significant (500cm) liver tissue constructs made of a new natural polymer composite, imbedded with optimized network of flow channels, and manufactured using innovative structured porogen and lost- ...
Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) condition... more Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.
Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as p... more Cumulative evidences suggest that nerve growth factor (NGF) promotes angiogenic effects such as proliferation and migration of endothelial cells (ECs) from different vascular beds, induces capillary sprouting in chorioallantoic membrane and improves in vivo vascularization in a hind-limb ischemic model. In the present study, we sought to investigate the signaling properties of NGF in a microcapillary ECs model compared to those of a neuronal model. NGF-induced phosphorylation of signaling molecules Erk1/2, Akt and PLCγ were measured using Western blotting and compared between mouse NGF (mNGF) and snake venom NGF analogues. NGFs-induced signaling was TrkA mediated as evident by inhibition with the TrkA antagonist K252a. NGF and its analogues-induced signaling in ECs were characterized by a transient effect in contrast to a prolonged stimulation in neuronal cells. The potency of mouse, cobra and viper NGFs to induce Erk1/2 phosphorylation in ECs was higher than in neurons. In ECs, mNGF exhibited the highest efficacy of stimulation of Erk1/2 phosphorylation, followed by viper and cobra NGFs. The efficacy of stimulation of Erk1/ 2 phosphorylation measured with neurons was opposite from that in ECs. NGF-induced temporal signaling differences between ECs and neurons may explain the dual vascular and neurotrophic effects of this growth factor.
Biochimica Et Biophysica Acta - General Subjects, Jun 1, 2015
Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The... more Cysteine-rich secretory protein (CRISP) is present in majority of vertebrate including human. The physiological role of this protein is not characterized. We report that a CRISP isolated from Echis carinatus sochureki venom (ES-CRISP) inhibits angiogenesis. The anti-angiogenic activity of purified ES-CRISP from snake venom was investigated in vitro using endothelial cells assays such as proliferation, migration and tube formation in Matrigel, as well as in vivo in quail embryonic CAM system. The modulatory effect of ES-CRISP on the expression of major angiogenesis factors and activation of angiogenesis pathways was tested by qRT-PCR and Western blot. The amino acid sequence of ES-CRISP was found highly similar to other members of this snake venom protein family, and shares over 50% identity with human CRISP-3. ES-CRISP supported adhesion to endothelial cells, although it was also internalized into the cytoplasm in a granule-like manner. It blocked EC proliferation, migration and tube formation in Matrigel. In the embryonic quail CAM system, ES-CRISP abolished neovascularization process induced by exogenous growth factors (bFGF, vpVEGF) and by developing gliomas. CRISP modulates the expression of several factors at the mRNA level, which were characterized as regulators of angiogenesis and blocked activation of MAPK Erk1/2 induced by VEGF. ES-CRISP was characterized as a negative regulator of the angiogenesis, by direct interaction with endothelial cells. The presented work may lead to the development of novel angiostatic therapy, as well as contribute to the identification of the physiological relevance of this functionally uncharacterized protein.
We investigated the effects of hypoxia on spontaneous (SP)-and activin A (AA)-induced definitive ... more We investigated the effects of hypoxia on spontaneous (SP)-and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O 2 , but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31-and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1a and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1a-/-mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1a-/-mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1a-/-mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.
Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoi... more Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and β1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ~5 μg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS.
Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) condition... more Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.
The intrinsic fluorescence of catecholamines can be exploited for a simple and speedy fluorescenc... more The intrinsic fluorescence of catecholamines can be exploited for a simple and speedy fluorescence assay of secretion from bovine adrenal chromaffin cells. Catecholamines are separated from concomitantly secreted proteins by centrifugation in 0.4 M perchloric acid, and their fluorescence is measured directly in a conventional spectrofluorometer without chemical derivatization. Catecholamine release, assayed by this technique, is virtually identical to data obtained in parallel by the classical trihydroxyindole method. Intrinsic catecholamine fluorescence does not differentiate between epinephrine and norepinephrine. Interferences from small molecular weight compounds not precipitated in perchloric acid are rare, and can be accounted for using appropriate calibration curves.
Anterior cruciate ligament (ACL) reconstructive surgery is a major health concern world-wide beca... more Anterior cruciate ligament (ACL) reconstructive surgery is a major health concern world-wide because of a large aging population and increased occurrence of sport-related damage. Tissue engineering is a rapidly growing interdisciplinary field that offers a promising new approach for ACL repair. In order to overcome the shortages of current existing surgical fixation devices, we are combining gradient cellular structure (GCS) injection molding technique and biomedical engineering to develop novel surgical fixation devices (screw, ...
Wolters Kluwer Health may email you for journal alerts and information, but is committed to maint... more Wolters Kluwer Health may email you for journal alerts and information, but is committed to maintaining your privacy and will not share your personal information without your express consent. For more information, please refer to our Privacy Policy. ... Skip Navigation Links Home > March/April 1997 - Volume 43 - Issue 2 > Exposing Endothelialized Cardiac Prosthesis to Pulsatile Flo... ... An abstract is unavailable. This article is available as a PDF only. ... Thought you might appreciate this item(s) I saw at ASAIO Journal. ... Your message has been successfully sent to your ...
Hemocompatibility of the blood-contacting surface of artificial cardiac prostheses remains a majo... more Hemocompatibility of the blood-contacting surface of artificial cardiac prostheses remains a major challenge for their long-term clinical use. We hypothesized that the thrombogenicity of segmented polyurethanes (PUS) used for creating the blood sacs can be reduced by lining the luminal surface with autologous endothelial cells (EC). Over the past years we have optimized our approach by 1. choosing an appropriate (detoxified) biomaterial (Biospan@), 2. generating a controlled, roughened PU surface and 3. precoating the luminal surface with an autologous protein complex (APC) made from plasma cryoprecipitate, 4. using autologous EC isolated from adipose tissue or the jugular vein, 5. designing a seeding device with 3-D rotation, which allows for the effective, uniform endothelialization of fully assembled cardiac prostheses,and 6. developing techniques for non-destructive monitoring of the extent of EC coverage. In vitro studies confirm the importance of exposing the EC lining the blood sacs to hemodynamic forces (flow and cyclic strain) prior to implantation for establishing a nonthrombogenic, shear-resistant EC monolayer. We recently tested fully endothelialized Ventricular Assist Devices (VADs) in vitro in a mock-loop circulation (3.2 l/min flow at 1 Hz) for 6 and 24 hours. At 6 hours, no denudation was observed. After 24 hours the integrity of the EC-monolayer on fibronectin-coated PUS was partially (<300/o) impaired. By contrast, virtually no denudation was observed when the cells were seeded on Biospan@ precoated with bovine cryoprecipitate. Similar results were obtained in our first er vivo tests in which fully endothelialized VADs were connected to an extracorporeal aorta-to aorta bypass in a calf model. Our results provide the first evidence for sucecssful in vitro and w vivo testing of endothelialized VADs and stress the importance of the composition of the extracellular matrix for maintaining a durable EC monolayer.
The Journal of Thoracic and Cardiovascular Surgery, 1995
Endothelial cell-lined in circulation skeletal muscle ventricles Skeletal muscle ventricles were ... more Endothelial cell-lined in circulation skeletal muscle ventricles Skeletal muscle ventricles were constructed from the latissimus dorsi in six dogs by wrapping the muscle around a polypropylene mandrel. Jugular vein endothelial cells were harvested enzymatically and grown in tissue culture. After 3 weeks of vascular delay and 4 weeks of electrical conditioning, five skeletal muscle ventricles were seeded with 5 to 8 x 106 autologous endothelial cells by percutaneous injection of a cellular suspension into the lumen of the skeletal muscle ventricle; one skeletal muscle ventricle was injected with culture medium alone as an unseeded control. The autologous endothelial cells were all prelabeled with a lipid-bound cellular marker, PKH-26. After an additional 4 weeks of electrical conditioning, the mandrels were removed and the skeletal muscle ventricles were connected to the descending thoracic aorta and activated to contract during cardiac diastole at a 1:2 ratio with the heart. After 3 hours of continuous pumping, mean diastolic pressure was increased by 35% (58-7 versus 78-6 mm Hg, p < 0.05). At this time, the skeletal muscle ventricles were excised for histologic examination. Sections stained with hematoxylin and eosin revealed a continuous cellular layer lining the skeletal muscle ventricle; no cells were present on the lumen of the control skeletal muscle ventricle. All seeded skeletal muscle ventricles exhibited fluorescence as a result of the PKH-26 cellular marker. Immunofluorescent staining with antibodies to von Willebrand factor and ultrastructural analysis with an electron microscope confirmed the endothelial character of these cells lining the lumen of the skeletal muscle ventricles. The ability to create endothelial cell-lined muscular pumping chambers holds important implications for the resolution of thrombotic events in cardiac assist devices as well as toward the clinical application of skeletal muscle ventricles. (J THORAC CARDIOVASC SURG 1995;109:66-73) Gregory A. Thomas, MD (by invitation), Peter I. Lelkes, PhD (by invitation), Susumu Isoda, MD, PhD (by invitation), Dawn Chick, BS (by invitation), Huiping Lu, MD (by invitation), Robert L. Hammond, BA (by invitation), Hidehiro Nakajima, MD, PhD (by invitation), Hisako Nakajima, MD (by invitation), Henry L. Waiters III, MD (by invitation), and
After nearly seven decades of development, dental composite restorations continue to show limited... more After nearly seven decades of development, dental composite restorations continue to show limited clinical service. The triggering point for restoration failure is the degradation of the bond at the tooth-biomaterial interface from chemical, biological, and mechanical sources. Oral biofilms form at the bonded interfaces, producing enzymes and acids that demineralize hard tissues and damage the composite. Removing bacteria from bonded interfaces and remineralizing marginal gaps will increase restorations' clinical service. To address this need, we propose for the first time the use of piezoelectric nanoparticles of barium titanate (BaTiO3) as a multifunctional bioactive filler in dental resin composites, offering combined antibacterial and (re)mineralization effects. In this work, we developed and characterized the properties of dental piezoelectric resin composites, including the degree of conversion and mechanical and physical properties, for restorative applications. Moreover, we evaluated the antibacterial and mineralization responses of piezoelectric composites in vitro. We observed a significant reduction in biofilm growth (up to 90%) and the formation of thick and dense layers of calcium phosphate minerals in piezoelectric composites compared to control groups. The antibacterial mechanism was also revealed. Additionally, we developed a unique approach evaluating the bond strength of dentin-adhesive-composite interfaces subjected to simultaneous attacks from bacteria and cyclic mechanical loading operating in synergy. Our innovative bioactive multifunctional composite provides an ideal technology for restorative applications using a single filler with combined long-lasting nonrechargeable antibacterial/remineralization effects.
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