Biomedical subjects

Dörthe Schmidt

Publications and source records attributed to Dörthe Schmidt.

11 recordsLinked to original sources

PubMed2006

Tissue engineered heart valves based on human cells.

Valvular heart disease is still a significant cause of morbidity and mortality worldwide. Clinically used valve replacements including mechanical valves as well as fixed biological xeno- or homografts are associated with several major disadvantages. Alternatively, tissue engineering aims at the fabrication of autologous living cardiovascular replacements with the potential to grow and to repair, particularly for paediatric applications. Therefore, autologous cells are harvested and seeded onto three-dimensional matrices followed by biomimetic in vitro conditioning enabling the development of the neo-heart valve tissue. Here, we review different human cell sources such as vessels, bone marrow, umbilical cord tissue and blood, and chorionic villi with particular regard to cell phenotypes and their suitability for extracellular matrix production for tissue engineering purposes.

Absorbable Implants↗

PubMed2006

Living autologous heart valves engineered from human prenatally harvested progenitors.

BACKGROUND: Heart valve tissue engineering is a promising strategy to overcome the lack of autologous growing replacements, particularly for the repair of congenital malformations. Here, we present a novel concept using human prenatal progenitor cells as new and exclusive cell source to generate autologous implants ready for use at birth. METHODS AND RESULTS: Human fetal mesenchymal progenitors were isolated from routinely sampled prenatal chorionic villus specimens and expanded in vitro. A portion was cryopreserved. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable leaflet scaffolds (n=12) and conditioned in a bioreactor. After 21 days, leaflets were endothelialized with umbilical cord blood-derived endothelial progenitor cells and conditioned for additional 7 days. Resulting tissues were analyzed by histology, immunohistochemistry, biochemistry (amounts of extracellular matrix, DNA), mechanical testing, and scanning electron microscopy (SEM) and were compared with native neonatal heart valve leaflets. Fresh and cryopreserved cells showed comparable myofibroblast-like phenotypes. Genotyping confirmed their fetal origin. Neo-tissues exhibited organization, cell phenotypes, extracellular matrix production, and DNA content comparable to their native counterparts. Leaflet surfaces were covered with functional endothelia. SEM showed cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves. CONCLUSIONS: Prenatal fetal progenitors obtained from routine chorionic villus sampling were successfully used as an exclusive, new cell source for the engineering of living heart valve leaflets. This concept may enable autologous replacements with growth potential ready for use at birth. Combined with the use of cell banking technology, this approach may be applied also for postnatal applications.

Absorbable Implants↗

PubMed2006

Functional growth in tissue-engineered living, vascular grafts: follow-up at 100 weeks in a large animal model.

BACKGROUND: Living autologous vascular grafts with the capacity for regeneration and growth may overcome the limitations of contemporary artificial prostheses. Particularly in congenital cardiovascular surgery, there is an unmet medical need for growing replacement materials. Here we investigate growth capacity of tissue-engineered living pulmonary arteries in a growing lamb model. METHODS AND RESULTS: Vascular grafts fabricated from biodegradable scaffolds (ID 18+/-l mm) were sequentially seeded with vascular cells. The seeded constructs were grown in vitro for 21 days using biomimetic conditions. Thereafter, these tissue-engineered vascular grafts (TEVGs) were surgically implanted as main pulmonary artery replacements in 14 lambs using cardiopulmonary bypass and followed up for < or = 100 weeks. The animals more than doubled their body weight during the 2-year period. The TEVG showed good functional performance demonstrated by regular echocardiography at 20, 50, 80, and 100 weeks and computed tomography-angiography. In particular, there was no evidence of thrombus, calcification, stenosis, suture dehiscence, or aneurysm. There was a significant increase in diameter by 30% and length by 45%. Histology showed tissue formation reminiscent of native artery. Biochemical analysis revealed cellularity and proteoglycans and increased collagen contents in all of the groups, analogous to those of native vessels. The mechanical profiles of the TEVG showed stronger but less elastic tissue properties than native pulmonary arteries. CONCLUSIONS: This study provides evidence of growth in living, functional pulmonary arteries engineered from vascular cells in a full growth animal model.

Absorbable Implants↗

PubMed2006

Activation of human microvascular endothelial cells with TNF-alpha and hypoxia/reoxygenation enhances NK-cell adhesion, but not NK-Cytotoxicity.

BACKGROUND: Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-alpha and hypoxia/reoxygenation in an in vitro model of I/R. METHODS: MVEC were stimulated in vitro for 8 h with TNF-alpha, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using Cr-release assays. RESULTS: Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-alpha but unaffected by hypoxia/reoxygenation in the absence of TNF-alpha. ICAM-1 expression was further increased by a combination of TNF-alpha and hypoxia/reoxygenation, whereas TNF-alpha-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-alpha and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-alpha and slighty reduced by hypoxia/reoxygenation. CONCLUSIONS: Endothelial activation with TNF-alpha, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.

Cell Adhesion↗

PubMed2006

Invited commentary.

Explore the source record for details and available documents.

Absorbable Implants↗

PubMed2006

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

BACKGROUND: Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. METHODS: Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). RESULTS: Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. CONCLUSIONS: These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

Biomechanical Phenomena↗

PubMed2005

Living patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells.

OBJECTIVE: A major shortcoming in contemporary congenital heart surgery is the lack of viable replacement materials with the capacity of growth and regeneration. Here we focused on living autologous patches engineered from human umbilical cord derived fibroblasts and endothelial progenitor cells (EPCs) as a ready-to-use cell source for paediatric cardiovascular tissue engineering. METHODS: EPCs were isolated from 20 ml fresh umbilical cord blood by density gradient centrifugation and myofibroblasts were harvested from umbilical cord tissue. Cells were differentiated and expanded in vitro using nutrient media containing growth factors. Before seeding, cell-phenotypes were assessed by immuno-histochemistry. Biodegradable patches fabricated from synthetic polymers (PGA/P4HB) were seeded with myofibroblasts followed by endothelialization with EPCs. All patches were cultured in a perfusion bioreactor. A subgroup of patches was additionally stimulated by cyclic strain. Analysis of the neo-tissues comprised histology, immuno-histochemistry, extracellular matrix (ECM) analysis and biomechanical testing. RESULTS: Endothelial phenotypes of EPCs before seeding were confirmed by Ac-Dil-LDL, CD 31, von-Willebrand-Factor and eNOS staining. Histology of the seeded patches demonstrated layered viable tissue formation in all samples. The cells in the newly formed tissues expressed myofibroblast markers, such as desmin and alpha-SMA. The EPCs derived neo-endothelia showed constant endothelial phenotypes (CD 31, vWF). major constituents of ECM such as collagen and proteoglycans were biochemically detected. Stress-strain properties of the patches showed features of native-analogous tissues. CONCLUSIONS: Living tissue engineered patches can be successfully generated from human umbilical cord derived myofibroblasts and EPCs. This new cell source may enable the tissue engineering of versatile, living, autologous replacement materials for congenital cardiac interventions.

Biocompatible Materials↗

PubMed2004

Umbilical cord blood derived endothelial progenitor cells for tissue engineering of vascular grafts.

BACKGROUND: A substantial limitation regarding present pediatric cardiac surgery is the lack of appropriate materials for the repair of congenital defects. To address this shortcoming, tissue engineering is a scientific field that aims at in vitro fabrication of living autologous grafts with the capacity of growth, repair, and regeneration. Here we focused on tissue engineered vascular grafts using human umbilical cord blood derived endothelial progenitor cells (EPCs), as a noninvasive cell source for pediatric applications. METHODS: EPCs were isolated from 20 ml fresh human umbilical cord blood by Ficoll gradient centrifugation and cultured in endothelial basal medium containing growth factors. After proliferation and differentiation cells were analyzed by immunohistochemistry and seeded onto three-dimensional (3D) biodegradable vascular scaffolds (porosity > 95%, n = 22). Twenty-four hours after seeding the vascular grafts were positioned into a pulse-duplicator-in vitro system and grown for 48 hours under biomimetic conditions. A second group was grown 6 days statically and an additional 6 days biomimetically. Controls were cultured statically. Analysis of the grafts included immunohistochemistry, histology, and scanning electron microscopy. RESULTS: Preseeding differentiated EPCs indicated constant endothelial phenotypes including acetylated low-density lipoprotein, cluster of differentiation 31, von Willebrand factor, and endothelial nitric oxide synthetase. Seeded EPCs established favorable cell-to-polymer attachment and proliferation into the 3D tubular scaffolds. Both conditioned and static cellular constructs demonstrated positive staining for cluster of differentiation 31, von Willebrand factor, and expression of endothelial nitric oxide synthase. CONCLUSIONS: Human umbilical cord derived EPCs indicated exceptional growth characteristics used for tissue engineering of vascular grafts. These cells demonstrated a constant endothelial phenotype and related functional features. Based on these results EPCs seem to be a promising autologous cell source with regard to cardiovascular tissue engineering, particularly for the repair of congenital defects.

Absorbable Implants↗

PubMed2003

Hypoxia and reoxygenation do not upregulate adhesion molecules and natural killer cell adhesion on human endothelial cells in vitro.

OBJECTIVES: Ischemia/reperfusion injury is characterized by endothelial cell activation leading to increased expression of adhesion molecules such as inter-cellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, endothelial- and platelet-selectin (E- and P-selectin), and to the subsequent recruitment of leukocytes. The aim of the present study was to investigate the respective effects of a proinflammatory cytokine (tumor necrosis factor alpha, TNF-alpha), hypoxia and/or reoxygenation on adhesion molecule expression and natural killer (NK) cell adhesion in an in vitro model of I/R. METHODS: Human aortic endothelial cells (HAEC) were stimulated in vitro for 8h with TNF-alpha (1000 U/ml) and exposed to hypoxia (1% O(2)), reoxygenation (21% O(2)) or different combinations thereof. Cell surface expression of ICAM-1, VCAM-1 and E-/P-selectin on HAEC was analyzed by flow cytometry, and culture supernatants were tested for soluble adhesion molecules by ELISA. Rolling adhesion of NK cells on HAEC was determined using a rotating assay. RESULTS: Untreated HAEC constitutively expressed ICAM-1 on their surface but neither expressed E-/P-selectin, VCAM-1, nor shedded soluble adhesion molecules. Exposure of HAEC to hypoxia or hypoxia and reoxygenation did not upregulate cell surface expression or shedding of adhesion molecules. In contrast, TNF-alpha significantly upregulated cell surface expression of ICAM-1, VCAM-1, and E-/P-selectin and led to the shedding of ICAM-1 and E-selectin. Combined treatment of HAEC with TNF-alpha, hypoxia and reoxygenation reduced E-/P-selectin surface expression and enhanced E-selectin shedding, but did not further influence ICAM-1 and VCAM-1. Soluble VCAM-1 was not detected. NK cell adhesion on HAEC increased 4-fold after TNF-alpha stimulation, but was not affected by hypoxia or hypoxia and reoxygenation. CONCLUSIONS: Both the expression of endothelial adhesion molecules and rolling NK cell adhesion was upregulated by TNF-alpha but not by hypoxia alone or hypoxia followed by reoxygenation supporting the view that anti-inflammatory treatment may reduce ischemia/reperfusion injury.

Aorta↗

PubMed2003

[Totally artificial training model for coronary heart surgery: the renunciation of animal experiments?].

AIM: Animal protection laws will lead to stricter and more selective criteria thus resulting in a decline of available animals. Yet to train cardiac surgical skills a totally artificial training model was developed. DESCRIPTION OF THE TRAINING MODEL: The model is based on differently hardened polyurethane. Cover is a 1:1 replica of the human thoracic wall. Disposable coronaries are integrated in the heart-model. Vessels and part of the ascending aorta can be rinsed. By means of a newly designed air-pump stroke volume, heart-rate and rhythm can be adjusted. EXPERIENCES: Set-up of the model is easy and quick. Accustomed instruments can be used. Handling of artificial tissue is nature-like. Degree of difficulty is dependent on stroke volume, heart rate, arrhythmia, vessel-size and vessel-quality. CONCLUSION: The phantom helps to achieve confidence in coronary revascularisation. It facilitates an accompanying training for the less-trained as well as the skilled surgeon. The nature-like characteristics will help to reduce animal experiments in future.

Animal Testing Alternatives↗

PubMed2002

NPC1: Complete genomic sequence, mutation analysis, and characterization of haplotypes.

Niemann-Pick type C disease (NP-C) is a rare, autosomal recessive lipid storage disorder. At least 96% of all NP-C patients link to NPC1 which encodes for a lysosomally-targeted protein. We describe the complete genomic sequence of 57,052 kb corresponding to the transcribed region of human NPC1 including several exonic and intronic single nucleotide polymorphisms (SNPs). Sequencing of all exons, splice sites, and the promoter region of NPC1 in 12 unrelated Caucasian NP-C patients revealed nine novel and four known most likely disease-causing mutations. Ten unique mutations found only once in 24 disease alleles were observed in patients being compound heterozygous for two different mutations. Two of the three missense mutations identified more than once were observed in a total of four patients homozygous for the respective mutation along with homozygosity for the underlying haplotype. The patients were offspring of most likely nonconsanguineous couples. Based upon genotyping exonic SNPs c.2572A>G (I858V; g.45020A>G) and c.2793C>T (N931N; g.45686C>T) and segregation analysis we characterized the haplotype of all 24 NPC1 alleles and of 138 alleles of healthy Caucasian control subjects. All four permutations between the two SNPs were identified in the control alleles: 2572A-2793C (50%), 2572G-2793T (41%), 2572G-2793C (5%), and 2572A-2793T (4%). These data are suggestive for an ancestral intragenic recombination within a genomic fragment of <666 bp. While 17 of 24 NP-C alleles (71%) shared haplotype 2572G-2793T, this haplotype accounted for only 41% in the controls (p=0.007; 2-sided Fisher exact test) suggesting the possibility of an influence of the haplotypic background on expression of missense mutations in NPC1.

Carrier Proteins↗