Gerhard Ziemer

Coating-techniques to improve the hemocompatibility of artificial devices used for extracorporeal circulation

OBJECTIVEnExtracorporeal circulation procedures have been shown to induce complement and leukocyte activation, release of endotoxin and inflammatory mediators, including cytokines, nitric oxide, oxygen free radicals, and platelet activating factors. The contact between the blood and the various artificial surfaces of the extracorporeal system results in an unspecific post-perfusion syndrome. For diminishing these negative side effects several coating-techniques have been developed to create devices with improved hemocompatibility.nnnMETHODSnThis review deals with the current knowledge of heparin-coated and otherwise surface-modified perfusion systems. The pathway how heparin-coated surfaces work is discussed and techniques for surface-coatings, both clinically introduced as well as newly developed are presented.nnnRESULTSnNumerous clinical studies compared heparin-coated versus non-coated circuits. Heparin-bonded devices showed lessened humoral and cellular activation, in particular a reduced complement activation with a reduced inflammatory post-perfusion syndrome. Also platelet protection and more favorable post-operative lung function are of particular note. Recent clinical trials demonstrated shortened hospital stays, less drainage bleeding, and reduced cerebral complications using heparin-coated oxygenation systems. The diminished expression of the leukocyte adhesion molecules CD 11b/c in CBAS devices points to a decreased activation of neutrophils. In addition, one research group found a reduced production of oxygen radicals. Heparin-bonding minimizes oxygenator failure by a significant reduced pressure gradient across the oxygenator, probably caused by decreased fibrin and platelet deposition at the hollow fiber surfaces. A meta analysis examined the impact of heparin-bonded systems on clinical outcomes and resulting costs. Using heparin-bonded circuits led to total cost savings from US

A New Technique for the Isolation and Surface Immobilization of Mesenchymal Stem Cells from Whole Bone Marrow Using High-Specific DNA Aptamers

1000 to 3000. Several authors demonstrated reduced blood loss and better clinical outcome by reduction of systemic heparinization and the employment of heparin-coated devices.nnnCONCLUSIONnAbove and beyond the long-term applications, routine heart operations have also markedly begun to utilize heparin-coated devices. This trend will assuredly continue in the coming years and is an important step toward higher hemocompatibility of blood-contacting surfaces in the ECC device. Heparin-coatings are merely the beginning of improved hemocompatibility for all materials that come into contact with human blood or tissues. Intelligent materials with almost completely physiological surfaces will be at the surgeons disposal within the next few years.

Hemocompatibility of heparin-coated surfaces and the role of selective plasma protein adsorption

Adult mesenchymal stem cells (aMSCs) are a stem cell population present in bone marrow, which can be isolated and expanded in culture and characterized. Due to the lack of specific surface markers, it is difficult to separate the MSCs from bone marrow directly. Here, we present a novel method using high‐specific nucleic acids called aptamers. Porcine MSCs were used as a target to generate aptamers by combinatorial chemistry out of a huge random library with in vitro technology called systematic evolution of ligands by exponential enrichment (SELEX). After cloning and sequencing, the binding affinity was detected using a cell‐sorting assay with streptavidin‐coated magnetic microbeads. We also used 12‐well plates immobilized with aptamers to fish out MSCs from the cell solution and a fluorescein isothiocyanate‐labeled aptamer to sort MSCs from bone marrow using high‐speed fluorescence‐activated cell sorting. The cells showed high potency to differentiate into osteogenic, as well as into adipogenic, lineages with typical morphological characteristics. Surface marker staining showed that the attached cells were CD29+, CD44+, CD45−, CD90+, SLA class I+, SLA DQ−, and SLA DR−. Compared with existing methods, this study established a novel, rapid, and efficient method for direct isolation of aMSCs from porcine bone marrow by using an aptamer as a probe to fish out the aMSCs. This new application of aptamers can facilitate aMSC isolation and enrichment greatly, thereby enhancing the rate of aMSC‐derived cells after in vitro differentiation for various applications in the emerging field of tissue engineering and regenerative medicine.

Preservation of intact adult rat photoreceptors in vitro: study of dissociation techniques and the effect of light.

Although several studies have shown that heparin-coated surfaces reduce the activation of both the complement system and the coagulation system, there is still inadequate understanding of the factors initiating and controlling blood activation at these surfaces. We investigated the adsorption profile of 12 common plasma proteins (and the platelet receptor CD41) to a heparin coating (Carmeda BioActive surface (CBAS)) compared to uncoated controls (PVC) by using an in vitro whole blood Chandler-Loop model. Surface bound proteins were studied kinetically by a direct ELISA technique. Western blots were performed on the SDS eluates in order to detect adsorbed cleavage products and denatured proteins. Changes in plasma levels of neutrophil activation markers, platelet activation, coagulation activation, complement activation and the inflammatory response were measured by conventional ELISAs. This study showed significant differences in adsorption patterns among the heparin-coated and the uncoated surfaces, notably for fibronectin, fibrinogen, C3 and high molecular weight kininogen (HMWK). The kinetic studies confirmed the results obtained from Western blots and indicated specific adsorption profiles of plasma proteins. We assume that at least some of the improved blood compatibility of the heparin-coated surfaces may be ascribed to the selective uptake and cleavage of plasma proteins.

New strategies for in vivo tissue engineering by mimicry of homing factors for self-endothelialisation of blood contacting materials.

The generation of single-stranded DNA (ssDNA) molecules plays a key role in the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process and numerous molecular biology techniques and applications, such as DNA sequencing, single-nucleotide polymorphism (SNP) analysis, DNA chips, DNA single-strand conformation polymorphism (SSCP) analysis and many other techniques. The purity and yield of ssDNA can affect the success of each application. This study compares the two ssDNA production methods, the strand separation by streptavidin-coated magnetic beads and alkaline denaturation and the lambda exonuclease digestion, in regard to the purity of generated ssDNA and the efficiency. Here, we demonstrate the considerable benefits of ssDNA production by lambda exonuclease digestion for in vitro selection of DNA aptamers. We believe that the generation of ssDNA aptamers using this method will greatly improve the success rate of SELEX experiments concerning the recovery of target-specific aptamers.

A new sealant for knitted Dacron prostheses: Minimally cross-linked gelatin

For years intensive research has been done to endothelialise vascular prostheses with autologous endothelial cells before implantation in patients. However, this procedure is extremely time-, labor- and cost-intensive and can be realized only in very few clinical cases. The discovery of circulating endothelial progenitor cells (EPCs) in 1997 brought new perspectives for the endothelialisation of blood contacting materials. Coating of synthetic graft surfaces with capture molecules for circulating EPCs mimics a pro-homing substrate for fishing out EPCs directly from the bloodstream after implantation. These cells with high proliferation potential can cover the graft with non-thrombogenic endothelium which maintains optimal haemostasis and minimize the risk of restenosis. In this review, different concepts are discussed to capture circulating EPCs on synthetic vascular grafts after implantation. We hypothesize that in vivo self-endothelialisation of blood contacting materials by homing factor-mimetic capture molecules for EPCs may bring revolutionary new perspectives towards future clinical application of stem cell and tissue engineering strategies.

Treatment of periprosthetic soft tissue infection of the groin following vascular surgical procedures by means of a polyvinyl alcohol-vacuum sponge system

There has been a resurgence of interest in the concept of presealing high-porosity knitted Dacron prostheses with an absorbable biologic material. Such a material should provide reliable porosity control, preferably reducing water porosity from 2000 ml/cm2/min to less than 50 ml/cm2/min. It should not interfere with the fibrous and vascular ingrowth that securely anchors the developing pseudointima. In previous studies, we have examined fibrin glue and two forms of aldehyde cross-linked insoluble collagen used as Dacron sealants. We concluded that delayed resorption of the sealant as seen with glutaraldehyde cross-linked insoluble collagen results in undesirable healing characteristics, particularly lack of adhesion between pseudointima and the luminal surface of a prosthesis. This study examines a new sealant. Soluble collagen (gelatin) is treated to reduce the number of free amino groups available for aldehyde cross-linking. It is then weakly cross-linked with an aldehyde mixture and applied to a knitted Dacron prosthesis. Water porosity studies have confirmed satisfactory porosity control. Both rat subcutaneous and canine circulatory implants for 6 months reveal relatively rapid and complete sealant resorption without undesirable modification of the normal healing process of knitted Dacron.

Autograft Reinforcement to Preserve Autograft Function After the Ross Procedure A Report From the German-Dutch Ross Registry

Deep groin infections after prosthetic vascular surgical procedures represent a serious complication of surgical practice. Septicemia and/or erosive hemorrhage can both be consequences. In this situation, removal of the graft appears to be the only option. However, if the infection is detected early (type Szilagyi III), local treatment to eradicate the infection could serve as an alternative. Twenty‐four patients with confirmed infection of the soft tissue adjacent to the prosthetic material in the groin were treated locally by implantation of a vacuum sponge system. Duration of this treatment was 2 weeks. All patients showed excellent tissue granulation of the wound area and the microbial stains were negative at the end of therapy. In 21 patients the wound could be primarily closed after explantation of the sponge. Three patients underwent open treatment because of a skin defect. After 12u2003months, the wounds had healed well in all patients. Histologic evaluation revealed a physiological healing process. Deep soft tissue infections of the groin adjacent to prosthetic vascular material (type Szilagyi III) can be treated effectively and safely with the vacuum sponge system. The treatment is inexpensive, easy to perform, and the initial vascular reconstruction can be preserved. (WOUND REP REG 2003;11:104–109)

Fibrin sealant, aprotinin, and immune response in children undergoing operations for congenital heart disease

Background— Autograft reinforcement interventions (R) during the Ross procedure are intended to preserve autograft function and improve durability. The aim of this study is to evaluate this hypothesis. Methods and Results— 1335 adult patients (mean age:43.5±12.0 years) underwent a Ross procedure (subcoronary, SC, n=637; root replacement, Root, n=698). 592 patients received R of the annulus, sinotubular junction, or both. Regular clinical and echocardiographic follow-up was performed (mean:6.09±3.97, range:0.01 to 19.2 years). Longitudinal assessment of autograft function with time was performed using multilevel modeling techniques. The Root without R (Root−R) group was associated with a 6× increased reoperation rate compared to Root with R (Root+R), SC with R (SC+R), and without R (SC-R; 12.9% versus 2.3% versus 2.5%.versus 2.6%, respectively; P<0.001). SC and Root groups had similar rate of aortic regurgitation (AR) development over time. Root+R patients had no progression of AR, whereas Root−R had 6 times higher AR development compared to Root+R. In SC, R had no remarkable effect on the annual AR progression. The SC technique was associated with lower rates of autograft dilatation at all levels of the aortic root compared to the Root techniques. R did not influence autograft dilatation rates in the Root group. Conclusions— For the time period of the study surgical autograft stabilization techniques preserve autograft function and result in significantly lower reoperation rates. The nonreinforced Root was associated with significant adverse outcome. Therefore, surgical stabilization of the autograft is advisable to preserve long-term autograft function, especially in the Root Ross procedure.

Endothelial progenitor cell capture stents — hype or hope?

OBJECTIVEnMost commercially available fibrin sealants contain aprotinin in doses of 1500 kallikrein inactivator units per milliliter. They are used in many operative disciplines. An elevated risk of hypersensitivity reactions exists at reexposure to aprotinin. Our aim was to examine the immunogenic potency of aprotinin as a fibrin sealant content.nnnMETHODSnWe investigated 49 children with operatively treated congenital heart disease. All patients received aprotinin only topically as contained in fibrin sealant. Serum samples were drawn preoperatively, 1 week, 2 weeks, 6 weeks, and approximately 1 year after operation. They were analyzed for aprotinin-specific immunoglobulin G antibodies with a standard enzyme-linked immunosorbent assay and a fluorescence enzyme immunoassay for aprotinin-specific immunoglobulin E antibodies.nnnRESULTSnAt 1 week, 2 weeks, 6 weeks, and 1 year, we found prevalences of 8% (2 of 26), 8% (2 of 24), 6% (3 of 49), and 0% for aprotinin-specific Immunoglobulin E, and for aprotinin-specific immunoglobulin G 8% (2 of 26), 17% (4 of 24), 39% (19 of 49), and 12% (5 of 41). The doses of aprotinin given did not differ significantly in antibody-negative and antibody-positive patients; no significant factors could predict the immune response.nnnCONCLUSIONSnOur findings show the existence of a subgroup of patients who had aprotinin-specific antibodies develop after topical aprotinin application. Any use of aprotinin must be carefully documented. If aprotinin use is planned in patients who previously underwent a surgical procedure, preexposure to aprotinin in any form must be sought to avoid unexpected anaphylactic reactions. The necessity itself and alternatives for aprotinin as a stabilizing agent in fibrin sealants merit consideration.