Development and Characterization of a Porcine Mitral Valve Scaffold for Tissue Engineering.

Decellularized scaffolds represent a promising alternative for mitral valve (MV) replacement. This work developed and characterized a protocol for the decellularization of whole MVs. Porcine MVs were decellularized with 0.5% (w/v) SDS and 0.5% (w/v) SD and sterilized with 0.1% (v/v) PAA. Decellularized samples were seeded with human foreskin fibroblasts and human adipose-derived stem cells to investigate cellular repopulation and infiltration, and with human colony-forming endothelial cells to investigate collagen IV formation. Histology revealed an acellular scaffold with a generally conserved histoarchitecture, but collagen IV loss. Following decellularization, no significant changes were observed in the hydroxyproline content, but there was a significant reduction in the glycosaminoglycan content. SEM/TEM analysis confirmed cellular removal and loss of some extracellular matrix components. Collagen and elastin were generally preserved. The endothelial cells produced newly formed collagen IV on the non-cytotoxic scaffold. The protocol produced acellular scaffolds with generally preserved histoarchitecture, biochemistry, and biomechanics.

[Viability and Particle Size of Fat Grafts Obtained with WAL and PAL Techniques]. / Vitalität und Partikelgröße bei der autologen Fetttransplantation mit der WAL und der PAL Technik.

BACKGROUND: Water jet-assisted liposuction (WAL) and power-assisted liposuction (PAL) are used for autologous fat grafting. This study analyses the viability and particle sizes of fat grafts obtained by these techniques. PATIENTS, MATERIAL AND METHODS: The WAL and PAL techniques were applied in 9 female patients in identical body regions. In order to analyse cell viability, fat grafts were tested via the WST-8 assay and DNA quantification immediately after liposuction. Furthermore, in order to determine particle size, an optically evaluable water-fat emulsion was analysed by macroscopic inspection and light microscopy. RESULTS: The WST-8 assay showed significantly lower extinction values (OD) for use of the WAL technique - corresponding to a lower metabolical activity - compared to PAL liposuction: WAL 1.85±0.56 OD, PAL 2.25±0.57 OD. The quotient of extinction values and cell DNA concentration determined by DNA quantification also indicated statistically significant differences between both systems of liposuction in favour of using power-assisted systems: WAL 0.061±0.023 OD/µg, PAL 0.083±0.029 OD/µg. On the other hand, microscopic and macroscopic analyses showed significantly greater diameters (d) for fat grafts obtained with the PAL technique than by WAL liposuction: dmakroWAL=0.8 mm and dmakroPAL=1.1 mm or, respectively, dmikroWAL 0.89 mm and dmikroPAL=0.93 mm. CONCLUSION: Power-assisted liposuction obtains fat grafts with a higher metabolical activity than water jet-assisted liposuction. A falsification of extinction values within the WST-8 assay due to diversity of the number of cells was eliminated by additionally implemented DNA quantification. According to the current scientific debate, the particle size of obtained fat grafts is also considered as an important criterion for the success of autologous fat grafting. For clinical use, one should favour techniques which provide the smallest and most viable fat grafts as possible. In our opinion, the significantly lower size of WAL particles compared to the higher viability of PAL grafts indicates a necessity of analysing viability as well as particle size in order to evaluate liposuction systems. Data solely about in vitro viability of fat grafts fail to offer a recommendation for the use of a specific technique.

Adipogenic differentiation of laser-printed 3D tissue grafts consisting of human adipose-derived stem cells.

Laser-assisted bioprinting (LaBP) allows the realization of computer-generated 3D tissue grafts consisting of cells embedded in a hydrogel environment. In this study, human adipose-derived stem cells (hASCs) were printed in a free-scalable 3D grid pattern by means of LaBP. We demonstrate that neither the proliferation ability nor the differentiation behaviour of the stem cells was affected by the LaBP procedure. Furthermore, the 3D grafts were differentiated down the adipogenic lineage pathway for 10 days. We verify by quantitative assessments of adipogenic markers that the 3D grafts resemble cell lineages present in natural adipose tissue. Additionally, we provide the proof that even pre-differentiated hASCs could be utilized for the generation of 3D tissue grafts. These results indicate that the biofabrication of living grafts resembling their complex native origin is within reach.

[Viability of autologous fat grafts harvested with the Coleman technique and the tissu trans system (shippert method): a comparative study]. / Vitalitätsvergleich von Fetttransplantaten welche mit der Coleman Technik und dem Tissu Trans System (Shippert Methode) gewonnen wurden.

Various methods for harvesting and refining autologous fat grafts have been described. One of the standard procedures, the Coleman technique, is based on manual aspiration to reduce the negative presssure and the centrifugation of the grafts. The Shippert technique uses automatic liposuction with reduced negative pressure and abstains from centifugation in order not to reduce viability of the graft by exposing it to centrifugal forces. This study intends to compare the viability of fat grafts processed with the above-mentioned methods.Fat grafts were obtained in 9 patients by using both the Tissu Trans system (Shippert technique) and the Coleman technique. To evaluate the impact of centrifugation forces, the grafts harvested with the Coleman technique were treated with standard adjustment of the centrifuge and also with doubled g-force. Viability of fat grafts was analysed with the WST-8 test and with annexin V/PI assay FACS analysis.The viability of fat grafts processed by the Coleman technique was significantly higher compared to the Shippert technique on applying the WST-8 test. Applying the annexin V/PI analysis, the viability of fat grafts was almost equal with both techniques. Whereas the fat grafts processed with the Tissu Trans system are injected without condensation, the grafts refined with the Coleman technique were concentrated 3 times by centrifugation compared to the primary liposuctioned graft volumes.The Coleman technique allows the preparation of a fat graft containing more viable cells than the Shippert technique. This is in part due to the condensation of the graft by centrifugation using the Coleman technique. The factor of condensation of the grafts harvested and refined with the Coleman technique exceeds the factor of increased fat graft viability in comparison to the Shippert technique. The Tissu Trans system is more than twice as fast and easier to use with a preferential use for large volume grafts like in breast augmentation, whereas the Coleman technique produces a more condensed graft, favouring it for fat grafting to the face where less volume is needed.

A biological alternative to alloplastic grafts in dialysis therapy: evaluation of an autologised bioartificial haemodialysis shunt vessel in a sheep model.

OBJECTIVES: To evaluate bioartificial haemodialysis access grafts in a sheep model with respect to patency and morphology. MATERIAL AND METHODS: Bovine internal thoracic arteries (n=28) were decellularised. Fourteen grafts (DC grafts) were directly implanted as cervical AV shunts, the remaining were re-seeded with endothelial cells (ECs) derived from blood samples of the later ovine recipient (EC grafts) first. Following simulated punctures and duplex ultrasound scans to determine patency, grafts were explanted for immunohistochemical characterisation after 3 and 6 months, respectively. DC grafts underwent biomechanical testing for compliance (C), suture retention strength (SRT), and burst pressure (BP) before (n=6) and after (n=6) implantation. RESULTS: Following 3 and 6 months, the majority of EC (n=6/6; n=6/7) and DC grafts (n=5/6; n=5/7) were patent and not relevantly stenosed (peak systolic velocity: EC grafts=76 cm s(-1)±4; DC grafts=77 cm s(-1)±5). Simulated haemodialysis punctures revealed significantly shorter bleeding times in all bioartificial grafts than in native jugular veins (P>0.001). Comparing native carotid arteries with DC grafts prior to and post-implantation, the latter differed significantly with respect to C (P>0.001; P=0.005), whereas only pre-implant DC grafts differed regarding BP (P=0.002); no differences were observed for SRT. Histology revealed complete endothelial surface coverage of EC, but not DC grafts. Furthermore, DC grafts exhibited areas of pronounced tissue calcification. CONCLUSION: The preclinical development of a bioartificial haemodialysis access graft with promising mechanical and morphological properties in a sheep model is feasible.

Laser printing of cells into 3D scaffolds.

One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are required. The two-photon polymerization (2PP) technique, used in this report for the fabrication of scaffolds, allows the realization of arbitrary 3D structures with submicron spatial resolution. Highly porous 3D scaffolds, produced by 2PP of acrylated poly(ethylene glycol), are seeded with cells by means of laser-induced forward transfer (LIFT). In this laser printing approach, a propulsive force, resulting from laser-induced shock wave, is used to propel individual cells or cell groups from a donor substrate towards the receiver substrate. We demonstrate that with this technique printing of multiple cell types into 3D scaffolds is possible. Combination of LIFT and 2PP provides a route for the realization of 3D multicellular tissue constructs and artificial ECM engineered on the microscale.

Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022.

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent of the Fpg-sensitive modifications were repaired within 2 h. Under conditions that induced 10 Fpg-sensitive modifications per 10(6) bp (six 8-oxoG residues per 10(6) bp), approximately 60 mutations per 10(6) cells were induced in the gpt locus of the AS52 cells. A rather similar mutation frequency was observed when a plasmid carrying the gpt gene was exposed to Ro19-8022 plus light under cell-free conditions and subsequently replicated in bacteria. Sequence analysis revealed that GC-->TA and GC-->CG transversions accounted for 90% of the base substitutions. A significant generation of micronuclei was detectable in AS52 cells exposed to the photosensitizer plus light as well.

Oxidative DNA damage induced by visible light in mammalian cells: extent, inhibition by antioxidants and genotoxic effects.

The extent of the indirect DNA damage generated in mammalian cells by visible light because of the presence of endogenous photosensitizers was studied by means of repair endonucleases. In immortalized human keratinocytes (HaCaT cells) exposed to low doses of natural sunlight, the yield of oxidative DNA base modifications sensitive to the repair endonuclease formamidopyrimidine-DNA glycosylase (Fpg protein) generated by this indirect mechanism was 10% of that of pyrimidine dimers (generated by direct DNA excitation). A similar yield of Fpg-sensitive modifications, which include 8-hydroxyguanine, was observed in primary keratinocytes. The relative yield of oxidative base modifications decreased at higher light doses, probably as a result of photodecomposition of the endogenous chromophore involved. For the three cell lines tested, viz. HaCaT cells, L1210 mouse leukemia cells and AS52 Chinese hamster cells, the yield of oxidative base modifications generated by a low dose of visible light appeared to be correlated with the basal concentrations of porphyrins in the cells. Induction of cellular porphyrin synthesis by pretreatment with 5-aminolaevulinic acid increased the light-induced oxidative damage in L1210 cells several-fold. In both induced and uninduced cells, the damage was inhibited by more than 50% in the presence of ascorbic acid (100 microM), while alpha-tocopherol and the iron chelator alpha-phenanthroline had no effect and beta-carotene even increased the damage. Even high doses of visible light did not significantly increase the numbers of micronuclei in L1210 cells or of gpt mutations in AS52 cells. The negative outcome can be fully explained by the photobleaching of the endogenous photosensitizers, which prevents the generation of sufficiently high levels of oxidative DNA damage. Therefore, the mutagenic risk arising from the indirectly generated oxidative DNA modifications induced by sunlight may be underestimated when results obtained at high doses are extrapolated to low doses or low dose rates.

DNA oxidation products determined with repair endonucleases in mammalian cells: types, basal levels and influence of cell proliferation.

Purified repair endonucleases such as Fpg protein, endonuclease III and IV allow a very sensitive quantification of various types of oxidative DNA modifications in mammalian cells. By means of these assays, the numbers of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine (8-oxoG), were determined to be less than 0.3 per 10(6) bp in several types of untreated cultured mammalian cells and human lymphocytes and less than 10 per 10(6) bp in mitochondrial DNA from rat and porcine liver. Oxidative 5,6-dihydropyrimidine derivatives sensitive to endonuclease III and sites of base loss sensitive to endonuclease IV or exonuclease III were much less frequent than Fpg-sensitive modifications. Here, we summarize our indications that all Fpg-sensitive modifications are recognized under the assay conditions and that on the other hand there is no artifactual generation of oxidative damage during the analysis. In addition, we show that the steady-state levels of Fpg-sensitive modifications in human lymphocytes and in two mammalian cell lines were higher in proliferating than in resting (confluent) cells. Only some of the Fpg-sensitive base modifications induced by various oxidants are 8-oxoG residues, as demonstrated for the damage under cell-free conditions. The percentage was dependent on the species ultimately responsible for the DNA damage and was approx. 40% in the case of hydroxyl radicals and peroxynitrite, 75% for type II photosensitizers (reacting via singlet oxygen) and only 20-30% in the case of type I photosensitizers such as riboflavin and acridine orange, which are assumed to react directly with the DNA.

Determination of steady-state levels of oxidative DNA base modifications in mammalian cells by means of repair endonucleases.

The alkaline elution technique in combination with various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) was used to quantify steady-state (background) levels of oxidative base modifications in various types of mammalian cells. In human lymphocytes the number of base modifications sensitive to Fpg protein, which include 8-hydroxyguanine, was 0.25 +/- 0.05 per 10(6) base pairs. Even lower levels (0.07 +/- 0.02 per 10(6) bp) were observed in HeLa cells. The numbers of sites sensitive to the other repair endonucleases were below the detection limit (0.05 per 10(6) bp). In a direct comparison, the background level of Fpg-sensitive modifications determined by alkaline elution was much lower than the background level of 8-hydroxydesoxyguanosine (8-oxodG) determined after enzymatic DNA hydrolysis by HPLC and electrochemical detection. However, the number of additional Fpg-sensitive modifications induced by a photosensitizer plus light was similar to the additional number of 8-oxodG residues determined by HPLC with electrochemical detection. This indicates that the enzyme assay does not systematically underestimate the number of lesions and points to an artefactual generation of 8-oxodG during DNA isolation and hydrolysis.

Cell cycle defect in connection with oxygen and iron sensitivity in Fanconi anemia lymphoblastoid cells.

Fanconi anemia (FA) is an autosomal recessive disorder involving progressive pancytopenia, skeletal malformations, and a predisposition to leukemia. The in vitro growth of FA fibroblasts is impaired, due to a defective G2 phase traverse of the cell cycle. Analyzing the cell cycle of lymphoid cell lines (LCLs) obtained from peripheral blood of FA patients by transformation with Epstein-Barr virus, we found a similar G2 phase defect, which was dependent upon the oxygen concentration. In addition, FA cells exhibited hypersensitivity toward cis-dichlorodiammineplatinum and mitomycin C, and moderate sensitivity toward trans-dichlorodiammineplatinum. FA cells, however, showed no elevated sensitivity toward paraquat, an intracellular generator of superoxide radicals, or cumene hydroperoxide, a model organic peroxide. Chelating iron with low concentrations of o-phenanthrolin improved cell proliferation and G2 phase transit of FA cells at 20% oxygen, but little at 5% oxygen. LCL cultures from healthy subjects were inhibited in their proliferation rate at all concentrations of o-phenanthrolin. Exposure to excess iron, on the other hand, was very toxic to FA cells at 20%, but less toxic at 5% oxygen. In conclusion, the FA mutation leads to a cell cycle defect, which is expressed in cultures of lymphoid cells from FA patients, and involves hypersensitivity toward bifunctional alkylating agents, oxygen, and iron.

Transcription factor NF-kappa B is activated by photosensitization generating oxidative DNA damages.

Reactive oxygen intermediates like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and, then, in the activation and replication of human immunodeficiency virus (HIV)-1 in human cells. Because H2O2 can be converted into the highly reactive OH. at various locations inside the cells, we started to investigate the generation of Reactive oxygen intermediates by photosensitization. This technique is based on the use of a photosensitizer which is a molecule absorbing visible light and which can be located at various sites inside the cell depending on its physicochemical properties. In this work, we used proflavine (PF), a cationic molecule having a high affinity for DNA, capable of intercalating between DNA base pairs. Upon visible light irradiation, intercalated PF molecules oxidize guanine residues and generate DNA single-strand breaks. In lymphocytes or monocytes latently infected with HIV-1 (ACH-2 or U1, respectively), this photosensitizing treatment induced a cytotoxicity, an induction of NF-kappa B, and a reactivation of HIV-1 in cells surviving the treatment. NF-kappa B induction by PF-mediated photosensitization was not affected by the presence of N-acetyl-L-cysteine while strong inhibition was recorded when the induction was triggered by H2O2 or by phorbol 12-myristate 13-acetate. Another transcription factor like AP-1 is less activated by this photosensitizing treatment. In comparison with other inducing treatments, such as phorbol 12-myristate 13-acetate or tumor necrosis factor alpha, the activation of NF-kappa B is slow, being optimal 120 min after treatment. These kinetic data were obtained by following, on the same samples, both the appearance of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in cytoplasmic extracts. These data allow us to postulate that signaling events, initiated by DNA oxidative damages, are transmitted into the cytoplasm where the inactive NF-kappa B factor is resident and allow the translocation of p50/p65 subunits of NF-kappa B to the nucleus leading to HIV-1 gene expression.

Visible light generates oxidative DNA base modifications in high excess of strand breaks in mammalian cells.

The DNA damage induced by visible light in L1210 mouse leukaemia cells was analysed by an alkaline elution assay with specific repair endonucleases. DNA single-strand breaks and DNA modifications sensitive to FPG protein (formamidopyrimidine-DNA glycosylase), endonuclease III and exonuclease III were quantified in parallel. The light-induced cellular DNA damage was found to consist of many base modifications sensitive to FPG protein, which most probably are predominantly 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) residues. Base modifications sensitive to endonuclease III are virtually absent. The yield of the FPG-sensitive base modifications is 10-fold higher than that of single-strand breaks plus AP sites (sites of base loss). The described ratios of the various modifications indicate that the damage most probably results from a reaction of DNA with singlet oxygen (type II reaction) or directly with an excited endogenous photosensitizer (type I reaction) and is not mediated by hydroxyl radicals. Experiments with cut-off filters indicate that wavelengths between 400 and 500 nm are responsible for most of the modifications. The FPG-sensitive base modifications are repaired efficiently (t1/2 approximately 1 h at 37 degrees C). This is perhaps why the light-induced DNA damage is apparently associated with only low mutagenicity.

DNA damage induced by photosensitizers in cellular and cell-free systems.

The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA damage induced by photosensitizers in the presence of visible light. Under cell-free conditions, chemically unrelated photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA damage. It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base loss (sensitive to exonuclease III or endonuclease IV) is low. Therefore the damage is markedly different from that induced by hydroxyl radicals. Mechanistically, the generation of the base modifications sensitive to FPG protein involves singlet oxygen in some, but possibly not all cases, as substituting D2O for H2O increases the reaction yield six-fold in the case of methylene blue, but only 1.4-fold in the case of acridine orange. In plasmids from Salmonella typhimurium strains treated with methylene blue or acridine orange plus light and from Escherichia coli strains treated with acridine orange or proflavin plus light, the same type of damage was observed as under cell-free conditions. In L1210 mouse leukemia cells exposed to acridine orange plus light, the numbers of modifications sensitive to FPG protein and exonuclease III were quantified, in addition to strand breaks, by a modified alkaline elution assay. Again, the number of base modifications sensitive to FPG protein was found to be several-fold higher than the number of strand breaks and sites of base loss. It has to be concluded that the DNA damage in the intact cells is not mediated by hydroxyl radicals or cellular nucleases, but by the same mechanism as operates under cell-free conditions with these agents.

Use of repair endonucleases to characterize DNA damage induced by reactive oxygen species in cellular and cell-free systems.

A number of repair endonuclease, viz. endonuclease III, formamidopyrimidine-DNA glycosylase (FPG protein), endonuclease IV, exonuclease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue, riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (gamma-rays) consists of approx. equal levels of base modifications. AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe(III)-nitrilotriacetate in the presence of H2O2 do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III-but not by those AP endonucleases which cleave at the 3' site-and probably represent AP sites oxidized at C-1'. The damage induced by Fe(III)-bleomycin plus H2O2 deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular nucleases, but by the same mechanism that is responsible for the cell-free DNA damage.