Burkhard Schlosshauer

To improve the clinical performance of vascular prostheses, which is inacceptably low for implants with small diameters (< 6 mm), biofunctionalization of synthetic implants by endothelialization has become a major, although still unreached, aim. In order to be able to recruit native endothelial progenitor cells (EPCs) to luminal implant surfaces from the blood stream, we generated monoclonal antibodies against the EPC-specific vascular endothelial growth factor receptor 2 (VEGFR-2). Employing the very efficient genetic immunization strategy, > 10 000 hybridoma clones were generated. Screening with various deletion mutants of VEGFR-2, 49 highly-specific monoclonal antibodies (mAbs) covering all seven Ig domains of VEGFR-2 were selected. mAb 9H10 was characterized in detail. Once immobilized on synthetic surfaces, mAb 9H10 allowed, within min, nearly 100-fold enrichment of VEGFR-2-expressing cells under continuous flow conditions. Cell trapping was cell-type specific and essentially not affected by competing VEGFR-2-negative cells. To exclude that the antibody would adversely modify receptor responses, four different in vitro assays were employed. Cell proliferation, angiogenic tube formation, acetylated low-density lipoprotein uptake and VEGFR-2 phosphorylation remained unaffected, suggesting that the antibody did not interfere with the receptor functioning of human umbilical vascular endothelial cells. The molecular and cellular characteristics make the selected monoclonal antibody a very promising tool for the biofunctionalization of vascular implants. Copyright © 2016 John Wiley & Sons, Ltd.

Lesions of the central nervous system elicit inflammatory responses that counteract the regeneration of neurites. Microglia and infiltrating macrophages that were activated by trauma have been identified as cellular sources of inhibitory factors. We examine cultured macrophage (RAW264.7) and neuronal (PC12) cell lines to ascertain the potential modulators of the inflammatory impact on neurons. By exposing quiescent macrophages to lipopolysaccharide (LPS) and interferon γ (IFN-γ), cells can be transformed into an activated M1 phenotype. Neurite extension was induced in PC12 cells by culturing them in the presence of nerve growth factor. Neurite outgrowth was quantified by analyzing immunofluorescence and phase contrast microscopy images. Activated macrophages significantly reduced neurite extension. Macrophage activation by LPS/IFN-γ induced a 1000-fold increase in tumor necrosis factor alpha (TNF-α) secretion, as quantified by enzyme-linked immunosorbent assays (ELISA). Recombinant TNF-α inhibited neurite formation at concentrations as low as 0.016 ng/ml. In contrast, the masking of TNF-α with specific functional antibodies abrogated neurite growth inhibition by activated macrophages. Taken together, these results indicated that TNF-α is a key component of inhibitory macrophage action. The transfection of PC12 neurons with microRNA-124 (miR-124) counteracted the inhibition of neurites mediated by both recombinant TNF-α and macrophages. miR-124 did not stimulate neurite formation per se, nor was cell viability affected. These data suggest that miR-124 might be a valuable tool for desensitizing neurons to a repulsive inflammatory environment.

In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin) to generate a blend (technical term: quattroGel), an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

Telomerase reverse transcriptase (TERT) is fundamental in determining the life span by regulating telomere length of chromosomes. To address the question whether the enhancement of the proliferative potential hampers cell differentiation, we generated TERT-over-expressing endothelial cells (ECs) and analyzed in vitro their (1) barrier function; (2) low-density lipoprotein uptake; (3) expression pattern of six selected marker proteins; (4) angiogenic potential in four assays; and (5) inflammatory responses. In contrast to investigations with focus on other cell parameters, we demonstrate that immortalization of ECs by over-expression of TERT resulted in different angiogenic and inflammatory behavior in comparison to cells with low native telomerase levels.

Microstructured 20 μm thick polymer filaments used as nerve implants were loaded with chitosan/siRNA nanoparticles to promote nerve regeneration and ensure local delivery of nanotherapeutics. The stable nanoparticles were rapidly internalized by cells and did not affect cell viability. Target mRNA was successfully reduced by 65−75% and neurite outgrowth was enhanced even in an inhibitory environment. This work, thus, supports the application of nanobiofunctionalized implants as a novel approach for spinal cord and nerve repair.

To more effectively manage the substantial bleeding encountered during surgical procedures in oto‐rhino‐laryngology, we developed a novel hemostatic sponge made of pharmaceutical grade, chemically cross‐linked gelatin. The sponge is characterized by a high pore density, reduced ligaments, and a high nanoscale roughness of lamella surfaces in the matrix. In vitro blood uptake assays revealed a very rapid absorption of human blood, which was two to three times faster than that measured with comparative hemostyptic devices. In an in vitro hemorrhage model using human veins, the novel gelatin sponge matrix induced hemostasis less than a minute after bleeding was induced. The sponge was shown to bring about rapid hemostasis when it was administered in a young patient suffering from acute bleeding of a pharyngeal angiofibroma, even though the patient had been treated with an anticoagulant because of a transient ischemic attack. As the gelatin matrix of the sponge is biocompatible and resorbable, the hemostyptic device could be left in place and was shown to be resorbed within 2 weeks. We hypothesize that the excellent hemostatic performance of the sponge might be linked to enhanced capillary effects in conjunction with optimized anchoring of fibrinogen on the nano‐rough material surface, as suggested by scanning electron microscopy. The novel gelatin sponge appears to be a promising hemostatic matrix, which could be of great benefit for patients suffering from epistaxis and other acute injuries resulting in severe bleeding. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2010.

By counteracting the shortening of chromosome telomeres, telomerase reverse transcriptase (hTERT) prevents senescence and age-related cell death. Embryonic cells display a high telomerase activity that declines rapidly with cell differentiation. Conversely, de-differentiated tumor cells tend to re-express telomerase. In view of the controversial data on the reciprocal correlation between cell proliferation and differentiation, we questioned whether telomerase overexpression and the resulting immortalization would affect the functional phenotype of human endothelial cells. Our comparative analysis addressed (1) distinct cell adhesion to different ECM-proteins analyzed on miniaturized multisubstrate arrays (MSA), (2) protein expression of diverse markers, (3) the uptake of DiI-Ac-LDL, (4) the inflammatory response based on upregulation of ICAM-1, (5) tube formation, and (6) the barrier properties of cell monolayers in transfilter cultures. Our results, based on some 40 data sets, demonstrate that immortalization of primary endothelial cells by hTERT maintains the typical endothelial characteristics without any sign of functional de-differentiation.

Lesions of the peripheral nervous system result in the loss of sensory and motor function and may in addition be accompanied by severe neuropathic syndromes originating from aberrant axonal regrowth. The transplantation of autologous nerve grafts represents the current "gold standard" during reconstructive surgery, despite obvious side effects. Depending on the demands of the lesion site, various donor nerves may be used for grafting (e.g., the sural, saphenous), sacrificing native functions in their target areas. Recently, several synthetic nerve guide implants have been introduced and approved for clinical use to replace autologous transplants. This alternative therapy is based on pioneering studies with experimental nerve guides. We present a comprehensive review of all published human studies involving synthetic nerve guides. Data from some 300 patients suggest that for short nerve defects of a few centimeters, resorbable implants provide promising results, whereas a number of late compression syndromes have been documented for nonresorbable implants. To treat longer defects, further implant development is needed, a goal that could be achieved, for example, by more closely imitating the intact nerve architecture and regulatory cell-cell interactions.

In a rat model, nerve regeneration was evaluated across a 2-cm defect in the median nerve using a resorbable artificial nerve conduit. The aim of this study was to develop an artificial, biocompatible, nerve guide to induce regeneration in the peripheral nervous system. The authors compared a nerve conduit of trimethylenecarbonate-co-epsilon-caprolactone (TMC/CL) filled with autologous Schwann cells with an empty hollow conduit and with an autologous nerve graft. Non-operated animals served as controls. The regeneration was evaluated with the grasping test, histological analysis of the nerve, muscle weight analysis (flexor digitorum sublimis muscle) and electrophysiological examination. After an observation period of 9 months, regeneration was seen only in animals that received an autologous graft or a Schwann cell containing nerve conduit. No signs of regeneration were found in animals supplied with the empty conduit. The results emphasize the important role of Schwann cells in the regeneration process across a 2-cm defect in the rat median nerve. Furthermore, the authors demonstrated that Schwann cell-filled nerve conduits induce functional recovery, as seen in the grasping test, that is comparable to that of the autologous graft 9 months after implantation.