Hans P. Wendel

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

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.

Induction of EPC homing on biofunctionalized vascular grafts for rapid in vivo self-endothelialization — A review of current strategies

For years intensive research has been done to improve the hemocompatibility of blood contacting vascular devices. Despite the enormous progress in physicochemical surface optimization technologies, the native endothelium still represents the ideal surface for blood contact. Numerous tissue engineering strategies aspired towards the endothelialization of graft surfaces to generate a non-thrombogenic barrier on artificial materials. A paradigm change in surface modification concepts is the in vivo endothelialization of vascular grafts by capturing circulating endothelial progenitor cells (EPCs) directly from the blood stream via biofunctionalized implant materials. Thereby, capture molecules are immobilized on artificial vascular grafts to mimic a pro-homing substrate for EPCs. In this review, different coating strategies for in vivo capturing of EPCs on synthetic implants are discussed. This therapeutic concept opens a new chapter in regenerative medicine by realizing the vision that every patient seeds his implants with his own progenitor cells to make the synthetic grafts unrecognizable for the bodys rejection mechanisms.

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

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.

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.

Platelets Recruit Human Dendritic Cells Via Mac-1/JAM-C Interaction and Modulate Dendritic Cell Function In Vitro

Objective—Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). Methods and Results—The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin &agr;M&bgr;2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C–dependent mechanism. Conclusions—Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

Thrombogenicity of Various Endovascular Stent Types: An In Vitro Evaluation

PURPOSE The aim of this study was to evaluate the thrombogenicity of different peripheral stent types in a standardized in vitro model with fresh human whole blood. MATERIALS AND METHODS Different stents (N = 77; n = 7 of each of 11 types) were implanted in polyvinyl chloride tubing loops and filled with donor blood samples. After 120 minutes of blood circulation, the thrombin-antithrombin III complex (TAT) levels, beta-thromboglobulin (beta-TG) levels, and platelet counts were assessed. RESULTS After 2 hours, significant differences were seen. TAT values (+/- SD) with the investigated stents were 31 micro g/mL +/- 20 (control, no stent), 328 micro g/mL +/- 206 (Saxx stent, peripheral medium CrNi31 L), 651 micro g/mL +/- 760 (Palmaz Corinthian Stent, 316 L stainless steel, electropolished), 1,609 micro g/mL +/- 1,264 (Palmaz Corinthian Stent, 316 L stainless steel, not electropolished), 810 micro g/mL +/- 578 (Palmaz Schatz long medium stent), 569 micro g/mL +/- 347 (Smart Nitinol stent), 1,037 micro g/mL +/- 577 (Megalink peripheral stent), 543 micro g/mL +/- 487 (peripheral stent, electropolished), 1,674 micro g/mL +/- 2,057 (peripheral stent, not electropolished), 3,128 micro g/mL +/- 1,812 (SelfX Nitinol stent, polished), 5,897 micro g/mL +/- 2,380 (SelfX Nitinol stent, unpolished), and 1,458 micro g/mL +/- 887 (bridge stent). The platelet count (x1,000/ micro L +/- SD) was 218 +/- 35 (control, no stent), 188 +/- 22 (Saxx stent), 187 +/- 20 (Palmaz Corinthian stent, electropolished), 135 +/- 37 (Palmaz Corinthian stent, not electropolished), 170 +/- 24 (Palmaz Schatz stent), 180 +/- 36 (Smart Nitinol stent), 159 +/- 26 (Megalink peripheral stent), 173 +/- 17 (peripheral stent, electropolished), 133 +/- 51 (peripheral stent, not electropolished), 123 +/- 37 (SelfX Nitinol stent, polished), 52 +/- 27 (SelfX Nitinol stent, unpolished), and 130 +/- 31 (bridge stent). CONCLUSION This standardized study showed a wide range of platelet activation after stent implantation. Electropolishing clearly reduced the thrombogenicity of the stents.

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

OBJECTIVE Most 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. METHODS We 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. RESULTS At 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. CONCLUSIONS Our 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.

Blood cell and plasma protein repellent properties of Star-PEG-modified surfaces

The implantation of biomaterials, medical devices or prostheses can instigate a rejection response or initiate an undesirable adsorption of plasma proteins, as well as blood cells on the implant surface, thus triggering diverse defense mechanisms against the supposed pathologic invader. The extent of this inflammatory reaction depends in part on the biocompatibility of the used materials or coatings. Although adsorption and coagulation responses can appear during the total in vivo lifetime of the implant, they are initially and crucially formed within the first 2–4 weeks of implantation. This early phase is of decisive importance for the consecutive in-growth and healing process. The present study was intended to elucidate the effects of blood contact to surfaces modified with reactive six-arm star-shaped poly(ethylene glycol-stat-propylene glycol) pre-polymers (Star PEG). Taken together, for Star-PEG-covered substrates we could demonstrate a profound reduction of various blood–biomaterial interactions compared to non-coated substrates, indicating the promising potential of this material as future coating for biomaterials with blood contact.

Endothelial progenitor cell capture stents — hype or hope?

The first report of late stent thrombosis leading to acute myocardial infarction in patients receiving the new CD34 antibody coated endothelial progenitor cell (EPC) capture stent may cast doubts on this technology. In our letter we review the potential risks and benefits of these stent types and give a short overview of recent alternative stent coatings for in vivo self endothelialization by using peptides, magnetic molecules or aptamers. Drawbacks with EPC capture stents should not lead to condemning this strategy in general, but rather stimulate research efforts to develop more suited capture molecules and more sophisticated coatings for the post-DES era towards more biological coatings which mimicry progenitor cell homing factors for rapid vascular wall regeneration.

Platelet derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro

Patients with myocardial infarction reveal an altered number of circulating mesenchymal stem cells (MSCs). Recently, it was shown that MSCs are able to regenerate myocardial tissue and to differentiate into endothelial cells. The homing mechanisms of MSCs from the circulation into the target tissue, however, are not understood so far. In this study, we evaluated the impact of platelets on MSC recruitment, proliferation, migration and integration into the endothelium. MSCs expressing alpha(v)beta(3) integrin were recruited to human arterial endothelial cells exposed to isolated platelets or IL-1 beta under high shear conditions. Furthermore, induction of vascular injury in vivo resulted in increased recruitment of injected MSCs as assessed by intravital microscopy and depletion of platelets significantly reduced this adhesion. The interaction of platelets and MSCs was inhibited by pre-incubation with the mAb 7E3 or an RGD protein both blocking beta(3) integrin mediated adhesion. Platelets had a chemotactic effect on MSCs, promoted a migratory MSC phenotype and dose- and activation-dependently enhanced migration of MSCs, a process, which was mediated by basic fibroblast growth factor (bFGF). Similarly, platelet derived bFGF increased proliferation of MSCs. Coincubation of MSCs with platelets facilitated integration into an endothelial monolayer, which was significantly reduced by pre-incubation with a blocking mAb to bFGF. We conclude that platelets may play a critical part in the recruitment of MSCs to the endothelium, influence MSC function and promote integration of MSCs into the endothelium.