The technology in cryotechnology.

The process of freezing biological material at extremely low temperatures is known as cryopreservation. To ensure the preservation of cells and tissues over an extended period of time, low temperatures are applied since biological processes, including the biochemical ones, come to a halt under cryogenic conditions and thus it is possible to maintain their structural and functional integrity. The field of cryopreservation gained more prominence in the 20th century and emerged as an unavoidable technology for different applications such as cell therapy, tissue engineering, or assisted fertilization. In this work we provide an overview of various technologies in the field of cryotechnology with regard to the freezing, storage and thawing of living cells. The first part covers the freezing process, starting with cryoprotective agents regarding their protection mechanisms and compositions, passing by cryo-imaging, micro-fluidic systems, and the currently available freezing and biobanking equipment. The second part focusses on the thawing process as well as the hypothermic preservation for the short-term storage of biological materials and constructs. Doi.org/10.54680/fr23610110112.

Contrast-enhanced nano-CT reveals soft dental tissues and cellular layers.

AIM: To introduce a methodology designed to simultaneously visualize dental ultrastructures, including cellular and soft tissue components, by utilizing phosphotungstic acid (PTA) as a contrast-enhancement agent. METHODOLOGY: Sound third molars were collected from healthy human adults and fixed in 4% buffered paraformaldehyde. To evaluate the impact of PTA in concentrations of 0.3%, 0.7% and 1% on dental soft and hard tissues for CT imaging, cementum and dentine-pulp sections were cut, dehydrated and stained with immersion periods of 12, 24 h, 2 days or 5 days. The samples were scanned in a high-resolution nano-CT device using pixel sizes down to 0.5 µm to examine both the cementum and pulpal regions. RESULTS: Dental cementum and periodontium as well as odontoblasts and predentine were made visible through PTA staining in high-resolution three-dimensional nano-CT scans. Different segments of the tooth required different staining protocols. The thickness of the cementum could be computed over the length of the tooth once it was made visible by the PTA-enhanced contrast, and the attached soft tissue components of the interior of the tooth could be shown on the dentine-pulp interface in greater detail. Three-dimensional illustrations allowed a histology-like visualization of the sections in all orientations with a single scan and easy sample preparation. The segmentation of the sigmoidal dentinal tubules and the surrounding dentine allowed a three-dimensional investigation and quantitative of the dentine composition, such as the tubular lumen or the ratio of the tubular lumen area to the dentinal surface. CONCLUSION: The staining protocol made it possible to visualize hard tissues along with cellular layers and soft tissues in teeth using a laboratory-based nano-CT technique. The protocol depended on both tissue type and size. This methodology offers enhanced possibilities for the concomitant visualization of soft and hard dental tissues.

Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

Cryobiological parameters of multipotent stromal cells obtained from different sources.

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × Vo) and bone marrow (0.28 × Vo) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol-1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me2SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol-1 and 27.4 ± 0.9 kcal mol-1 for Am and BmMSCs respectively. In the presence of Me2SO, activation energies were found to be 11.6 ± 0.3 kcal mol-1 and 19.5 ± 0.5 kcal mol-1 respectively. Furthermore, Me2SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to -30 °C, followed by 3 °C/min to -80 °C.

Effects of cryopreservation on the epigenetic profile of cells.

Effective cryopreservation protocols are essential for long-term storage of cells and their subsequent clinical application. Freezing protocols are generally considered as safe; however, putative effects on epigenetic marks have not yet been studied in detail. While post-thaw cell survival rates have been used to evaluate the success of cryopreservation protocols, increasing evidence suggests that freezing may be associated with deviations from the physiological epigenetic marks with putative long-term effects on the cells and/or their derivatives. A better understanding of the underlying mechanisms would be beneficial for improving safety and effectiveness of freezing protocols. The purpose of this review is to provide current information regarding epigenetic alterations (DNA methylation and histone modification patterns) associated with cryopreservation.

Active control of the nucleation temperature enhances freezing survival of multipotent mesenchymal stromal cells.

Cryopreservation is a technique that has been extensively used for storage of multipotent mesenchymal stromal cells (MSCs) in regenerative medicine. Therefore, improving current cryopreservation procedures in terms of increasing cell viability and functionality is important. In this study, we optimized the cryopreservation protocol of MSCs derived from the common marmoset Callithrix jacchus (cj), which can be used as a non-human primate model in various pathological and transplantation studies and have a great potential for regenerative medicine. We have investigated the effect of the active control of the nucleation temperature using induced nucleation at a broad range of temperatures and two different dimethylsulfoxide concentrations (Me2SO, 5% (v/v) and 10%, (v/v)) to evaluate the overall effect on the viability, metabolic activity and recovery of cells after thawing. Survival rate and metabolic activity displayed an optimum when ice formation was induced at -10 °C. Cryomicroscopy studies indicated differences in ice crystal morphologies as well as differences in intracellular ice formation with different nucleation temperatures. High subzero nucleation temperatures resulted in larger extracellular ice crystals and cellular dehydration, whereas low subzero nucleation temperatures resulted in smaller ice crystals and intracellular ice formation.

Dimethyl sulfoxide and ethylene glycol promote membrane phase change during cryopreservation.

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to study the effects of dimethyl sulfoxide and ethylene glycol on cell pellets of human pulmonary microvascular endothelial cells during freezing from 4 degree C to -60 degree C at 1 degree C per min. FTIR analysis showed that membranes undergo a phase change in the presence of cryoprotective agents (CPAs) which was not observed in the absence of CPAs. Cryomicroscopy revealed the formation of intracellular ice and concomitant cell volume changes. Intracellular ice was detected in the majority of the cells both in the presence and absence of CPAs. Membrane phase changes were found to be most pronounced at intermediate concentrations of cryoprotective agents; for dimethyl sulfoxide at around 1 M and for ethylene glycol at around 1.5 M. At those concentrations cell survival after thawing exhibited a maximum. The results indicate that CPAs promote rather than prevent cell dehydration during freezing.

Impact of valve calcification on systolic and diastolic valvular function--an in vitro model.

AIM: Despite continuous development of anticalcification treatment for bioprosthetic valves, calcification remains one major cause of structural failure. The aim of this study is to investigate changes in hemodynamic performance and leaflet kinematics in progressively calcified pericardial and porcine aortic valve prostheses. METHODS: Five pericardial (Edwards Perimount Magna) and 5 porcine (Medtronic Mosaic Ultra) aortic valve prostheses (Ø23 mm) were exposed to a high concentration Calcium-phosphate fluid in an in vitro pulse duplicator (300 cycles/minute) for 6 weeks. The prostheses were removed weekly and tested in an artificial circulation system (70 beats/min, Cardiac Output 5 l/min). All prostheses underwent X-ray, computed tomography (CT)-Scan and photographic examination for evaluation of progressive calcification. Leaflet kinematics were visualized with a high-speed camera. RESULTS: Pericardial valves demonstrated faster degeneration with significantly larger radiographic areas of leaflet calcification (16.5+/-4.3% versus 5.6%+/-2.0%) and also significantly higher Ca-uptake (170+/-71 microg/mg versus 103+/-49 microg/mg) after 6 weeks. Despite degeneration systolic function remained superior for pericardial valves (mean effective orifice area [EOA] 1.52+/-0.05 versus 1.28+/-0.11 cm2, P<0.01), but leaflet kinematics showed longer closing times (135+/-11 msec versus 85+/-9 msec after 6 weeks) accompanied by higher regurgitant flow (7.8+/-1.12 mL versus 1.2+/-0.28 mL, P<0.001). CONCLUSION: In vitro pericardial valves calcified faster and more severe than porcine valves leading to impaired diastolic function with prolongation of closing times and higher closing volume. Systolic function remained almost undisturbed by the calcification process. As a consequence in clinical settings, follow-up examinations for structural valve deterioration in porcine valves should focus on systolic performance, in pericardial valves on diastolic function.

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.

Dynamic in vitro calcification of bioprosthetic porcine valves: evidence of apatite crystallization.

OBJECTIVE: Calcification is the most important cause of structural deterioration of glutaraldehyde-fixed bioprosthetic valves. Devitalization of tissue favors calcium deposits in the shape of apatite crystals. Host factors influence the extent and progression of calcification, but the phenomenon can also occur in vitro in the absence of a viable milieu. Whether calcific deposits obtained in vitro are similar to those found in vivo is unknown. METHODS: Four porcine frame-mounted bioprostheses (St Jude Medical Bioimplant; St Jude Medical, Inc, St Paul, Minn) were tested in vitro by using a pulsatile accelerated calcification testing device at a frequency of 300 cycles per minute at 37 degrees C for 19 x 10(6) cycles with a rapid synthetic calcification solution (final product [calcium x phosphate], 130 mg/dL(2)). Three of the same type of xenografts explanted from human subjects because of calcific failure (time in place, 108 +/- 25.63 mo) served as control grafts. Each sample underwent gross and x-ray examination, histology, transmission and scanning electron microscopy, atomic absorption spectroscopy, electron microprobe analysis, and x-ray powder diffraction methods. RESULTS: All in vitro bioprostheses were heavily calcific, with intrinsic Von Kossa stain-positive deposits and a mean calcium content of 205.285 +/- 64.87 mg/g dry weight. At transmission electron microscopy, nuclei of calcification involved mostly collagen fibers and interfibrillar spaces and, more rarely, cell debris and nuclei. Electron microprobe analysis showed a Ca/P atoms ratio of 4.5:3, a value intermediate between hydroxyapatite and its precursor, octacalciumphosphate. X-ray powder diffraction showed a well-separated and sharp peak, which is typical of hydroxyapatite. Aggregates of plate-like crystals up to 8 microm in size were observed at scanning electron microscopy, with a typical tabular hexagonal shape consistent with apatite. The morphologic and chemical findings in human explants were similar. CONCLUSIONS: Intrinsic calcification of glutaraldehyde-fixed porcine valves was induced in vitro. Electron microprobe analysis and x-ray powder diffraction findings were in keeping with apatite crystallization, such as that occurring in valve xenografts implanted in vivo. The model may be of value to accelerate the screening of anticalcific agents and may reduce the need for animal experiments.

In vitro testing of bioprostheses: influence of mechanical stresses and lipids on calcification.

BACKGROUND: Structural valve deterioration of bioprostheses is mainly caused by the progressive development of calcification. Mechanical stresses or lipid deposits in porcine aortic leaflets have been proposed as major factors contributing to the calcification process. METHODS: A new test protocol consisting of nondestructive holographic interferometry, which allows a quantitative deformation analysis of heart valves, and accelerated dynamic in vitro calcification was used. The rapid calcification fluid contained a final combined calcium and phosphorus concentration of 130 (mg/dL)2 in barbital buffer solution. The calcification of 32 bioprostheses donated by different manufacturers (SJM Bioimplant, Biocor standard, Biocor No-React, Carpentier-Edwards SAV, Bravo, pericardial prototype) was assessed after up to 25 x 10(6) cycles by microradiography and the areas of calcification were compared with the holographic interferograms. The distribution of lipid droplets of four porcine prostheses were visualized by Sudan III stain before the calcification process. RESULTS: Most of the tested bioprostheses had areas presenting with stress concentrations, and the dynamic in vitro testing resulted in leaflet calcification corresponding to the holographic irregularities. A strong correlation between calcification and stress distribution or lipid accumulation was found (r = 0.72; r = 0.81, respectively). After 19 x 10(6) cycles, the Carpentier-Edwards SAV and the pericardial valves had significantly less calcification than other prostheses tested (p = 0.003), but the variation among individual prostheses from the same manufacturer was even more pronounced. CONCLUSIONS: Mechanical stresses or lipid accumulation seems to play an important role in the calcification process of bioprostheses. Quality control of bioprosthetic valves using holographic interferometry has the potential to predict calcification before implantation.

In vitro calcification of pericardial bioprostheses.

BACKGROUND AND AIM OF THE STUDY: The lifetime of bioprosthetic heart valves is limited by calcification. To investigate the calcification behavior of bioprostheses and gain insight into the etiology of valve calcification, a test protocol for accelerated valve calcification was developed. This protocol includes a pulsatile valve tester, a synthetic calcification fluid, and non-destructive radiographic assessment of calcification sites. About 40 porcine bioprostheses from different manufacturers have been investigated previously using this test protocol and showed that valves exhibited different calcification patterns and even different degrees of calcification within their leaflets. A positive correlation of calcification versus tissue anomalies/stress concentrations (r = 0.72; n = 29 valves) and lipid deposits (r = 0.81) was found. In the present study, bovine pericardial valves were investigated in comparison with porcine valves. METHODS: Four bovine pericardial and two porcine mitral valves (Baxter) with a tissue annulus diameter (TAD) of 29 mm (one 27 mm) were investigated in parallel under identical test conditions. The valves were cyclically loaded at 300 per min with a delta p of 110 mmHg at 37 degrees C for up to 19 x 10(6) cycles. The synthetic calcification fluid was changed weekly. Sites of calcification were assessed by microradiography. Radiographs were analyzed by PC images processing with respect to the degree of calcification, defined as calcified surface area in relation to total leaflet surface area. RESULTS: This analysis showed that, for bovine pericardial valves, the mean degree of calcification increased by 14% and 20% after 12 and 19 x 10(6) cycles, respectively. Under identical conditions, the mean degree of calcification of porcine valves increased by 28% and 37%. CONCLUSIONS: Pericardial valves appear less prone to calcification than porcine valves. Further studies must be performed in order to prove this finding since, as recognized previously in porcine valves, other factors such as tissue or manufacturing anomalies may be as important as the tissue source itself.

Holographic interferometry and in vitro calcification: comparing pericardial versus porcine bioprostheses.

BACKGROUND AND AIMS OF THE STUDY: Structural valve deterioration of bioprostheses is mainly caused by progressive calcification. It has not yet been convincingly demonstrated whether pericardial or porcine bioprostheses are more prone to calcification. METHODS: A previously described in vitro test protocol consisting of non-destructive holographic interferometry, which permits quantitative deformation analysis of heart valves and accelerated dynamic calcification in vitro was used to evaluate five stented pericardial bioprostheses of different sizes and design (three or two leaflets) from one manufacturer. The extent of calcification was assessed after up to 20 x 10(6) cycles in the valve tester by microradiography, and areas of calcification were compared by holographic interferometry using computerized image processing. Calcification was confirmed by EDX-analysis and Von Kossa staining. Results were compared with in vitro testing of 25 porcine bioprostheses from different manufacturers. RESULTS: The tested pericardial bioprostheses had an individual distribution of mechanical stresses detectable by holographic interferometry, which resulted in different calcification of valve leaflets. A strong correlation between calcification and stress distribution was found (correspondence of affected areas: 82.3 +/- 10.1%, r = 0.97). Variability in calcification and stress distribution, respectively, of pericardial valves compared well with our findings for porcine prostheses. Overall, the extent of leaflet calcification was not statistically different for pericardial and porcine bioprostheses (p = 0.21). CONCLUSIONS: The biological material of bioprostheses (pericardial versus porcine) does not seem to be the crucial factor in the calcification process. Mechanical stresses detectable by holographic interferometry have a more pronounced impact and predict calcification of individual prostheses, at least in the in vitro setting.

Quality control of bioprosthetic heart valves by means of holographic interferometry.

BACKGROUND AND AIMS OF THE STUDY: Limited durability of porcine bioprostheses is mainly caused by the progressive development of calcification. We tested the hypothesis that hidden tissue anomalies or unfavorable stress concentrations of commercially available bioprostheses may lead to later calcification and dysfunction. Application of holographic interferometry for non-destructive testing of biological heart valves enables a full-field analysis of heart valves and reveals deformation irregularities of valve tissue. MATERIAL AND METHODS: We developed an accelerated calcification protocol for bioprosthetic heart valves including an accelerated pulsatile valve tester for simultaneous testing of 10 heart valves under identical conditions and a rapid synthetic calcification fluid containing a final Ca x P of 130 (mg/dl)2 in barbital buffer solution. Ten porcine bioprostheses (St. Jude Medical, Bioimplant) were assessed by holographic interferometry and subjected to the pulsatile accelerated calcification process. Distribution and amount of calcification was evaluated by microradiography after 12 x 10(6) and 19 x 10(6) cycles, respectively. Areas of irregular fringe patterns detected by holography as well as areas of calcification were calculated and compared using a personal computer. RESULTS: All tested bioprostheses had localized or extended areas with holographic irregularities and the accelerated valve testing protocol resulted in even macroscopically visible calcifications at various sites. Comparative analysis of the obtained microradiographs revealed that 74.2% +/- 6.0% of calcified leaflet areas lay within the previously detected holographic anomalies. CONCLUSIONS: Our first results show a strong correlation between holographic anomalies and calcification of porcine bioprostheses. We conclude that suitable methods for evaluation and quality control of bioprosthetic heart valves are available and seem to be predictive with regard to valve calcification.

Synthesis, physical properties and preliminary investigation of hemocompatibility of polyurethanes from aliphatic resources with castor oil participation.

The synthesis of polyurethanes (PURs) from oligoetherdiol, two low molecular diols, castor oil and 4,4'-Methylenebis(cyclohexylisocyanate) is described. These polymers are characterized by measurements of the mechanical bulk and surface properties, preliminary investigation of compatibility with human blood and calcification in static conditions. The critical surface energy of synthesized PURs is similar to the critical surface energy of natural surfaces. Material-induced hemolysis and the changes of platelet counts in blood samples after contact with PURs are very low. Static seven-weeks-calcification testing in a synthetic calcification fluid did not indicate calcification by optical density measurements and by visual inspection and computer image processing of the X-ray films for PURs with and without castor oil.

In vitro blood damage by high shear flow: human versus porcine blood.

Devices for modern heart support are minimized to reduce priming blood volume and contact area with foreign surfaces. Their flow fields are partly governed by very high velocity gradients. In order to investigate blood damage, porcine and human blood was passed through a narrow Couette type shear gap applying defined high shear rates within the typical range for devices such as blood pumps or artificial heart valves (gamma = 1800/s to 110,000/s for 400 ms). Traumatization profiles of both blood species were recorded in terms of hemolysis and platelet count. Sublethal damage in terms of platelet (PF4) and complement activation (C5a) was additionally measured for human blood. Results for porcine and human blood were very similar. Hemolysis was not started until critical shear rates of about 80,000/s. Impact on platelets was severe with drops in cell count of up to 65% (at gamma = 55,000/s to 110,000/s) likely to set stronger limits to the design layout of devices than hemolysis. Concentrations of PF4 and C5a clearly increased with shear rate exhibiting stronger gradients where hemolysis started. Due to the similar results of porcine and human blood for hemolysis and platelet drop, porcine blood seems to be suitable for device testing. Selection of blood species would thus depend on handling, availability and analysis demands.

A new in vitro test method for calcification of bioprosthetic heart valves.

To investigate the calcification behavior of different bioprosthetic heart valves and verify possible hypotheses of the etiology of valve calcification, an accelerated pulse tester for bioprostheses was developed, whereby up to ten valves can be tested under identical test conditions. Each valve was mounted in a separate compartment on a piston and cyclically moved through a calcifying solution at frequencies of up to 800/min at 37 degrees C: An appropriate calcifying solution was evaluated by incubation tests of bovine and porcine tissue. Calcification was confirmed by measuring Ca and phosphate depletion by atomic absorption spectroscopy, von Kossa staining, EDAX, and microradiography. The first tests were successfully carried out on porcine valves that had been nondestructively assessed for tissue/stress anomalies by holographic interferometry prior to the calcification test. The tests showed that 75% of irregular fringe pattern areas corresponded to the calcification areas.

Thermophysical properties of a monolayer tissue with respect to freeze-drying.

Conservation of tissue structures by means of freeze-drying is still limited as the complex mechanisms taking place at molecular/cellular level are not fully understood. The successful application of hydroxyethyl starch (HES) in combination with maltose, sucrose, and trehalose as stabilizers of lipid bilayers/membranes in red blood cells suggests an extended use of this mixture of cryoprotectants. The effectiveness of such cryoprotectant solutions has been linked to changes in the thermophysical properties of cellular structures. This work deals, in a first step, with the thermophysical properties of a model monolayer tissue--onion epidermis--in binary aqueous solutions of dissacharides. First and second order phase transitions, i.e. melting, crystallisation, and glass transition, are characterised by means of Modulated Differential Scanning Calorimetry (MDSC).

Preparation of collagen scaffolds and their applications in tissue engineering.

Freeze-dried collagen scaffolds can be used for a variety of medical tissue engineering applications. The pore structure of the scaffolds might play a decisive role for the inoculation, growth and differentiation of the cells. For a controlled 3D-cell growth the pore structure needs to be homogeneous and the pore size individually adjustable. For lyophilised scaffolds, the pore structure is determined by the ice crystal morphology during freezing under steady conditions. Scaffolds with a homogeneous pore structure and a range of pore size between 25 and 100 microns were reached. Cells such as preadipocytes, keratinocytes, and fibroblasts showed to adhere well to the collagen matrix.

Endothelialization of electrospun polycaprolactone (PCL) small caliber vascular grafts spun from different polymer blends.

Small caliber vascular grafts represent a challenge to material scientists. In contrast to large caliber grafts, prostheses with diameter <6 mm, lead to increased hemodynamic disturbances and thrombogenic complications. Thus, endothelialization of small caliber grafts should create a compatible interface for hemodynamic processes. The purpose of our study was to compare different compositions of electrospun scaffolds with conventional ePTFE grafts with an inner diameter of 4 mm as well as different pre-coatings to create an optimized physiological interface for endothelialization. Polycaprolactone, polylactide, and polyethylenglycol (PCL/PLA and PCL/PLA/PEG) electrospun grafts and ePTFE grafts were pre-coated with blood, gelatine or fibronectin and seeded with endothelial cells from the human term placenta. Best results were obtained with fibronectin-coated PCL/PLA/PEG grafts. Here, the number of attached viable cells was 78-81% higher than on fibronectin pre-treated ePTFE grafts. Cells attached to PCL/PLA/PEG grafts appeared in physiological cobblestone morphology. Viability analysis showed a high cell viability of more than 98%. Fibronectin-coated PCL/PLA/PEG grafts may be a promising improvement to conventionally used ePTFE grafts.