BioStar 2008–Science in Exchange Meeting Abstracts

Plenary opening lecture

Session I: Regenerative BiologyKeynote Lecture

Session II: Regenerative TherapiesKeynote Lecture

Session III: InterfacesKeynote Lecture

Session IV: Medical Biology

Session V: Stem Cell NichesKeynote Lecture

Poster Presentations

PP 58 Characterization of Endothelial Progenitor Cells (EPC) in Healthy Volunteers

Introduction: Endothelial Progenitor Cells (EPCs) and their potential to differentiate into mature endothelial cells remain of high interest in regenerative medicine. EPCs are primitive cells found in peripheral blood and bone marrow. Published literature concerning the characterization of EPCs however is heterogenous and inconsistent. Objective of this study is to perform a comparing characterization of EPC using flow‐cytometry (FACS) and conventional assays for colony forming units (CFUs).

Material and Methods: A total of 18 healthy male Caucasian volunteers (23 to 43 years) were studied. Peripheral blood samples were taken 4 times in 2 weeks (day 1, 4, 8, 11). An EPC‐CFU‐Hill assay was performed as well as flow‐cytometrical analysis for CD34, KDR, CD133 and with CD45 as a negative exclusion.

Results: A characteristic CFU‐morphology was found for each volunteer but the number of CFU's varied between test days strongly (averaged: 10 ± 10 /1.9cm2). By flow cytometrical analysis we found about 0.004 ± 0.002 % (% of PBMCs) KDR‐positive cells, 0.10 ± 0.05 % CD34‐positive and 0.12 ± 0.03 % CD133‐positive cells. A strong correlation was found for CD34‐ and CD133‐positive signals. KDR and CD133 as well as KDR and CD34 correlated less.

Conclusion: CFU‐asays are not exclusive for EPCs. FACS analysis, in contrast enable a clear characterization of EPCs. No correlation was found between the number of CFUs and FACS data. Based on this findings future EPC studies should be conducted by FACS analysis.

PP 59 Transpericardial infusion of BBMC in acute Infarction

Cardiovascular illnesses continue to be the first cause of death in Brazil. Stem cells can be applied on treating cardiovascular diseases because its capability to induct neovascularization and tissue formation. Stem cells transplantation has been applied through the intracoronary, intramyocardic and retrograde infusion techniques. We present the transpericardic technique as an alternative route for infusion of a total of 108 bone marrow mononuclear (BMMC) in 6 female health swine submitted to acute myocardic infarction (AMI) using a balloon catheter under fluoroscopy. Injection of BMMC was also done in animals free of infarct for control group. The procedures were done in accordance to the School's Bioethics Committee under the n° 750/2005. Bone marrow (BM) was sample from the iliac crest using a bone punction needle and heparinized syringes.

BMMC were separated from the BM by density gradient using Ficoll‐Paque™ Plus (Amsersham Pharmacia) and stained with Hoechst 33342 (Invitrogen). After MAI occurrence, the epicardic space was accessed by pre umbilical region at median portion using an epidural needle to inject the BMMC. Ventricles were sliced into three portions and overlaid by a scintillographic grid to determine echocardiographic regions and to collect 17 fragments of each one, and evaluated by standard histology and immunehistochemistry. Total number of BMMC in the free area and of infarct in the ventricle was 109494 ± 46505 cells/area. We believe our study show low costs, easy‐maneuver, fast application of the technique and characterizes the epicardic space as a long‐term source of infusioned cells in the cardiac therapy.

PP 60 Same retention of mononuclear bone marrow cells after intracoronary infusion with or without balloon in infarcted pig hearts

Objectives: In clinical studies on cell‐therapy for acute myocardial infarction (MI), cells are usually applied by intracoronary infusion with balloon (IC/B). To test the utility of balloon‐occlusion, mononuclear bone‐marrow cell (MNC) retention after intracoronary infusion without balloon (IC/noB) was compared with IC/B and intramyocardial injection (IM).

Methods and Results: Four hours after LAD‐ligation in male pigs, reperfusion was allowed (confirmed by coronary angiography). Five days later, 1 × 10⁸ autologous ¹¹¹Indium‐labelled MNC were injected IC/noB (n = 4), IC/B (n = 4), or IM (n = 4). After 1h the fraction of injected MNC that was detected in the heart was 4.1 ± 1.1% in IC/noB, 6.1 ± 2.5% in IC/B (P = 0.19), and 20.7 ± 2.3% in IM (P < 0.001 vs. IC/noB and vs. IC/B). After 24h it was 3.0 ± 0.6% in IC/noB, 3.3 ± 0.5% in IC/B (P = 0.43), and 15.0 ± 3.1% in IM (P < 0.001 vs. IC/noB and vs. IC/B). Dynamic scintigrammes during IC/B showed a rapid 19.6 ± 8.0% cell‐loss during balloon‐inflation (no‐flow period) and a rapid 36.6 ± 17.8% cell‐loss after balloon‐deflation (re‐flow period). After IC/noB, the peak cell‐deposit was lower, followed by one phase of rapid cell‐loss (30.9 ± 11.0% in 6min). After IM, a slow linear cell‐loss was observed (9.7% per h). In histology, PKH‐67‐labelled MNC only rarely had passed the endothelial barrier at 24h after IC, while they were exclusively found in the interstitium after IM.

Conclusions: The observation of a similar cell‐persistence after intracoronary injections with and without balloon‐occlusion suggests that the balloon‐procedures currently applied in clinical studies are not necessary for cell‐deposit. If longer‐term cell‐persistence plays a role in cardiac cell‐therapy, IM may be superior to IC applications.

PP 61 Survival and tumor formation of undifferentiated embryonic stem cells injected into infarcted mouse hearts is dose‐Dependent

Objectives: Embryonic stem cells (ESC) can differentiate into all cardiac tissues and therefore are suitable for cardiac cell therapy. We investigated the influence of the injected cell number and of immunosuppression on survival and tumor formation after injection of undifferentiated ESC into cryo‐infarcted mouse‐hearts.

Methods: 10⁵ or 10⁶ undifferentiated male ESC were injected into the peri‐cryo‐infarct region (2 × 5μl) of female mice. Half of the animals additionally received immunosuppression with Cyclosporin‐A (+CyA). After 0h, 48h, 5d, 3w and 6w heart, lungs, spleen, kidneys, muscle, brain, liver and blood were explanted. The number of surviving (male) ESC was determined by real‐time PCR for y‐chromosome and tumors were identified by histology.

Results: After injection of 10⁵ ESC without immunosuppression, male cells were detectable in the heart only until 5d, whereas at 6w numbers had increased 20‐fold in 10⁵ + CyA, 20‐fold in 10⁶, and 30‐fold in 10⁶ + CyA, leading to highest numbers of male cells in 10⁶ (P < 0.01 vs. 10⁵) and 10⁶ + CyA (P < 0.001 vs. 10⁵ and vs. 10⁵ + CyA). This tumor formation was accompanied by a higher mortality (P of chi‐square < 0.05) and by a higher distribution of male cells to other organs than the heart at 6w after intramyocardial injection (P of chi‐square < 0.05).

Conclusions: Survival, proliferation and tumor formation of undifferentiated ESC in cardiac cell therapy depends on the number of transplanted ESC and on immunosuppression. There seems to be a critical cell number that is completely eliminated by the recipient's immunosystem while proliferation and tumor formation is unlimited with higher cell numbers and boosted by immunosuppression.

PP 62 The effect of application of G‐CSF and a CD26‐Inhibitor on stem cell homing and cardiac function after myocardial infarction

Background: The key issue of therapeutic stem cell approaches emerges to be the process of cardiac homing of stem cells via the SDF‐1‐CXCR4 axis. Myocardial SDF‐1, is degradeted by the extracellular protease CD26. We hypothesized that inhibition of CD26 by Diprotin A improves the homing of G‐CSF‐mobilized stem cells after myocardial infarction in a mouse model.

Methods: We induced myocardial infarction (AMI) in 8‐12 weeks old male C57BL/6 mice using surgical occlusion of the left descending artery. Mice were then treated either with G‐CSF (100 μg/kg/d i.p.) in combination with Diprotin A (140 μg /kg/d μg s.c., “G‐CSF+DipA”), G‐CSF (“G‐CSF”) alone or saline (“control”). FACS analyses were used to determine stem cell populations in peripheral blood and myocardium after 6 days. Cardiac function parameters were assessed using a Millar‐Tip catheter system. Survival was analyzed by Kaplan‐Meier‐method for 30 days (n = 20 in each group).

Findings: In G‐CSF+Diprotin A treated mice, cardiac homing of CD34+CXCR‐4 stem cells was significantly improved. This led to a relevant induction of resident cardiac stem cells (CD34‐CD45‐Sca1+ and CD34−CD45−ckit+) in the G‐CSF+DipA group compared to G‐CSF alone. Cardiac regeneration was improved in G‐CSF+DipA mice as shown by a significant improvement of left ventricular ejection fraction after 30 days. Finally, G‐CSF+DipA mice showed a significant increase of survival after 30 days.

Interpretation: This is the first study showing that combined application of G‐CSF and a CD26‐inhibitor improves cardiac function and survival after myocardial infarction by an enhanced cardiac homing of stem cells due to inhibition of SDF‐1 degradation.

PP 63 3D co‐culture construct of endothelial cells and cardiomyocytes; first steps towards a vascularized artificial myocardial tissue

Background: A remaining problem in generating 3D artificial tissues is the efficient supply of cells with nutrients and oxygen. Our approach is to vascularise myocardial tissue in vitro by an engineered vessel system in biological vascularised matrix (BioVaM). This study concentrates on the generation of an artificial cardiac construct based on a co‐culture of neonatal rat cardiomyocytes (nRCm) and endothelial cells derived from human cord blood endothelial progenitor cells (hCBEC) on BioVaM.

Methods: A co‐culture medium was established for cultivation of hCBEC and nRCm, by testing several media in respect to their impact to cell morphology, proliferation and differentiation. Differentiation of cells was evaluated by expression of specific markers for both cell types (PCR). Vessel structures of dezellularized segments were reseeded with 1.6 × 10⁷ TAMRA labeled hCBECs. Submucosa was reseeded with CSFE labeled 1 × 10⁵ nRCm/cm² one day later. nRCm sheets were stacked onto nRCm monolayers on submucosa at 3d. Constructs were examined after 7d.

Results: RPMI supplemented with 10% FCS and EGM‐2 Single‐Quots (Lonza) showed best results for both cell types. Proliferation rate was comparable to each specific media. Cell morphology stayed well defined and the state of differentiation was preserved. Constructs started to contract in a defined contraction direction following orientation of the collagen fibers on submucosa. Fluorescence microscopy revealed nRCm layers parallel aligned to the longitudinal axis, as well as reseeded vessel structures underneath.

Conclusion: Our construct may represent a first step towards the generation of a complex artificial vascularized cardiac construct for the therapy of injured myocardium.

PP 64 CCN1 enhances reendothelialisation in tissue engineered vascular constructs

Introduction: A major problem in generating 3D‐artificial myocardial tissue is an efficient supply of oxygen and nutrients to cells in vitro. Recently we reported the generation of an in vitro vessel‐bed based on biological vessel structures for vascularization of artificial tissues. Here we show the improvement of the reendothelialisation process by coating vessel structures with the proangiogenic CCN1.

Methods: Porcine small intestine segments with preserved pedicles were decellularised. Vessel structures were incubated with 100ng/mL CCN1 in culture media for 12h and reseeded with 1.6 × 10⁷ TAMRA labeled human cord blood derived endothelial cells (hCBEC). Constructs with (cBioVaM) and without (BioVaM) CCN1 were cultivated for 4d (n = 6) or 14d (n = 6). The outcomes were quantified by investigation of DNA content and lactate concentration. Fluorescence microscopy was performed to detect seeded cells inside the constructs. Findings were qualified using an arbitrary rating system. Endothelial character of cultivated cells was investigated using Dil‐Ac‐LDL‐assay.

Results: DNA content, measured 12 h after reseeding procedure, was increased 3× in cBioVaM compared to BioVaM. Increasing colonisation of vessels was observed by TAMRA fluorescence microscopy for all constructs. Quantification revealed an elevated increase of 70% after 4d and 50% after 14d cultivation in cBioVaM compared to BioVaM. Lactate was 2.6× higher in cBioVaM than BioVaM after 4d and 6.9× higher after 14d. Dil‐Ac‐LDL assay revealed endothelial character of seeded hCBEC in all constructs.

Conclusion: Coating of decellularised vessel structures with CCN1 supports the adhesion and reendothelialisation of BioVaM. Such constructs may solve the problem of nourishing cells inside 3D‐artificial constructs.

PP 65 Electrical integration promotes maturation of transplanted cardiomyocytes

Electrical integration and maturation of transplanted immature cardiomyocytes are essential to enhance functional benefit and reduce arrhythmogenic risk. Yet, little is known about the time course of integration and maturation.

Methods: Fetal cardiomyocytes (12.5‐15.5 days p.c.) expressing eGFP were transplanted into cryoinjured non‐transgenic adult mouse hearts. At 6, 9 and 12 days after transplantation, viable tissue slices of recipient hearts (thickness 150 μm) were prepared and intracellular action potentials of transplanted and host cardiomyocytes within the slices were recorded by sharp glass electrodes. Analyzed cells were marked by tetramethylrhodamine injections via the recording pipette, transplanted cells were identified by their green/red dual fluorescence. Slices were stimulated by a unipolar electrode placed in healthy host tissue.

Results: In transplanted cells coupled to healthy host myocardium, action potential duration at 50% repolarization (APD50), which decreases during native murine development, decreased from 32.2 ± 3.3 ms (day 6) to 27.9 ± 2.6 ms (day 9) and 19.6 ± 1.6 ms (day 12). The latter value was equal to the APD50 of host cells (20.5 ± 3.2 ms, P = 0.78), whereas cell size of transplanted cardiomyocytes was considerably smaller compared to host cardiomyocytes. The maximal stimulation frequency without conduction blocks, which was regarded as indicator of the quality of electrical integration of transplanted cells, was inversely proportional to the APD50 at 2 Hz (r² = 0.59). In transplanted cells embedded in the cryoinjury, which showed no electrical integration, APD50 was 59.2 ± 4.3 ms at day 12.

Conclusions: Electrical maturation of transplanted cardiomyocytes is a time‐dependent process, depending on the quality of electrical integration.

PP 66 Improved vascular regeneration and impeding of re‐stenosis by Shikonin based stent coatings

Cardiovascular diseases are one of the most common diseases in our society. When infarcts occur one state of the art treatment is to remove the stenotic plaque and to graft a stent in the affected vessel area to maintain the blood‐flow. A severe side‐effect from vascular interventions applying stents are occurring re‐stenosis where the vessel lumen is constricted again by strongly proliferating cells, mainly smooth muscle cells and fibroblasts. These cells migrate from the intima to the intraluminal side and are respd are responsible for the late‐lumen‐loss and thus the occurrence of re‐stenosis. Studies with drug‐eluting‐stents came to the result that anti proliferating agents (e.g. sirolimus) show best effects in reducing the late‐lumen‐loss but preventing also the endothelial layer from proliferation and so inhibit the rehabilitation of an intact vascular wall. In search of a selectively acting drug we focused on a pharmaceutical called Shikonin. Shikonin which is a red solid substance isolated from the roots of Lithospermum erythrorhizon has many different modes of action. Recent studies demonstrated potent effects like anti‐tumoral, antimicrobial and antifungal features as well as antiviral ones. Our investigations revealed a dose‐dependant anti‐proliferating impact of Shikonin. Shikonin inhibits proliferation of smooth muscle cells and fibroblast but not endothelial cells. Hence Shikonin is a potent candidate for an application on stents through it's selective acting on the responsible cells for re‐stenosis. The received data from the in‐vitro experiments are actually verified in an animal model. When previous data is validated a valuable tool is generated for the cardiovascular treatment.

PP 67 Isolation and Expansion of Adult Cardiac Stem Cells from Rhesus Monkey

Recently resident cardiac stem cells have been identified in human and murine hearts. Messina et al. have reported the isolation of adult cardiac stem cells (CSCs) from biopsy samples, forming self‐adherent clusters termed “Cardiospheres”. Due to their high proliferative capacity, they could provide sufficient cell numbers for regenerative therapies using autologous cells. We were now able to isolate a corresponding cell type from atrial and ventricular biopsy specimens of Rhesus monkeys. The tissue was cut into pieces, treated with 0.2% trypsin and plated onto fibronectin‐coated dishes. Outgrowing cells were harvested by trypsination after 1‐3 weeks and were grown on poly‐D‐lysin coated culture surfaces in a low‐serum medium supplemented with B27, EGF, bFGF, cardiotrophin and thrombin. These culture conditions resulted in sphere formation similar to human CSCs. Like the human CSCs, Rhesus monkey CSCs express stem cell (ABCG2) and endothelial markers (flk‐1, vWF) and appear to have the properties of adult cardiac stem cells (GATA4, FOG2, Tbx20) as determined by RT‐PCR and/or immunofluorescence analysis. They are capable of long‐term self‐renewal for up to 40 passages. However, karyotype analysis of one long‐term culture revealed extensive chromosomal abnormalities; therefore, genetic stability has to be tested before therapeutic application. As described for human CSC, we did not observe efficient cardiac differentiation into spontaneously contracting cells in vitro; the regenerative effect of Rhesus CSC in vivo after transplantation in infarcted areas of SCID mice hearts is currently under investigation. These cells could potentially be used in a large animal model investigating stem cell mediated regeneration.

PP 68 Isolation, Characterization and Cryopreservation of neonatal, porcine Cardiomyocytes

Background: The development of artificial tissues become more and more important in the field of regenerative medicine, particularly in the usage as test systems. The prerequisite therefore are isolated primary cells with a high purity, viability and functionality after seeding on an appropriate matrix. Porcine cardiomyocytes are easier available than human`s and bear close physiological resemblances to human cardiac cells.

Methods: Our principle of isolation is based on perfusion with cold calcium‐free collagenase solution with the aid of an electrical pump. Over 60 minutes the enzyme solution passed through the myocardium with a constant pressure of 80 mmHg. Then the muscle was minced mechanically and a physiological cellular calcium concentration was re‐established in three steps with a incubation period of 10 minutes after each addition. After centrifugation and resuspension with medium (DMEM, 10 % fetal bovine serum (FBS), 1 % penicillin‐streptomycin (PS)) the cardiomyocytes were seeded in coated tissue culture flasks. For cryopreservation the fresh isolated cells were resuspended in DMEM, 10 % FBS, 1 % PS und 10 % DMSO and freezed at − 80°C. After one day the transfer to liquid nitrogen took place.

Results: Immediately after isolation more than 90% vital, rod‐shaped and spontaneous contracting cells were detected with trypan blue stain. Five days later all vital cells showed complete adhesion. The detached cardiomyocytes were verified with connexin43 –antibody and started to contract spontaneously again between day ten und fourteen after seeding. The thawed cells showed a marginal lower vitality and the same characteristics as fresh isolated cells.

PP 69 In Vitro Fascia Model for Hernia Research

Annually 200,000 inguinal hernia and 60,000 incisional hernia are diagnosed in Germany. By congenital or acquired weak points of the abdominal wall the egression of visceral organs is possible, which can successfully be treated meanwhile by different surgical operation procedures depending upon kind and extent of the hernia. To the accepted factors of risk for the hernia development belong patient‐specific influences of the genetics, environment, nutrition and comorbidities. The weighting of these factors is very likely different with the emergence of inguinal and incisional hernia and is therefore the subject of the scientific discussion. For the design of an in vitro fascia model primary fibroblast‐like cells from porcine fascia or human inguinal and incisional hernia are isolated. Risk markers for favouring the emergence of hernia are identified by immuno histological and molecular biological characterisation of the matrix and isolated cells. Additionally cells are cultivated in a collagen matrix, set under physiological conditions like breathing by a bioreactor system. The established system is to be used on the one hand as mechanistical study model. On the other hand as bioartificial tissue structure applications as test systems for new biological materials and medicinal products seems to be possible, which could be applied for testing new hernia therapy forms.

PP 70 Investigations on the biocompatibility of a new collagen‐Based matrix for human urothelial cells

In vitro engineering of lower urinary tract tissue equivalents suitable for reconstructive surgery might require biomaterials as cell carriers. Matrices have to be biocompatible, induce tissue regeneration, and must be subject to rapid degradation in vivo. Aim of the pilot study was to prove adherence, viability, and growth pattern of human urothelial cells (HUCs) seeded on a new factory‐made bovine collagen I‐based matrix. HUCs isolated from ureter tissue were labelled with the fluorescent cell linker PKH26, seeded onto the collagen matrix, and cultivated in serum‐free medium. Cell adherence was indirectly ascertained by counting cells in culture supernatant. Growth behaviour was studied by phase contrast microscopy and cryosections of the populated matrix. Viability of HUCs was analysed with the WST‐1 assay. HUCs grown on the collagen matrix were as homogeneously spread as HUCs seeded onto standard plastic surface. At day 1 after seeding the fraction of non‐adherent HUCs was slightly increased (2.2%) compared to the controls (0.3%), whereas at day 3 both groups revealed similar rates (0,4% and 0,3%, respectively). Viability of HUCs growing on the matrix revealed 111% of the control group at day 3. The cell‐matrix constructs could be easily detached from the culture dish and were manageable with surgical instruments. The data demonstrate a good in vitro biocompatibility of the new collagen matrix. We conclude that the matrix might be well suitable for construction of cell‐matrix implants for reconstructive surgery of the lower urinary tract. Further experiments with urothelial multilayers grown on the matrix will be performed.

PP 71 Human mesenchymal stem cells for treatment of urinary incontinence in a nude rat model

Urinary incontinence is associated with damaged or decreased sphincteric myofibers. Bone marrow‐derived mesenchymal stem cells (MSCs) have the capacity to differentiate into muscle cells. Study aim was to examine myogenic differentiation of human MSCs and to monitor their integration into the urethral sphincter in a nude rat model. To induce myogenic differentiation, human MSCs in P1 were exposed to 5‐azacytidine (5‐AZA) for 24 hours. Expression of smooth muscle alpha‐ and alpha/gamma‐actin, skeletal muscle MyoD, and slow muscle myosin was examined in differentiation‐induced MSCs (P2‐P6) and native MSCs (P0‐P6) by immunocytochemistry. PKH26‐labeled MSCs were directly injected into the bladder neck of athymic rats and monitored histologically after 4, 8, 12, 28, and 56 days. Native MSCs expressed alpha‐actin, alpha/gamma‐actin, and skeletal muscle MyoD in P0 in 62%, 60%, and 73%, resp. and in P1 in 94%, 100%, and 92%, resp.; MSCs in P3‐P6 only expressed alpha‐actin (65‐84%). In MSCs exposed to 5‐AZA alpha‐actin, alpha/gamma‐actin, and skeletal muscle MyoD were identifed in P2‐P6 in 86‐97%, 81‐100% and 63‐100%, resp. There was no reactivity for skeletal slow muscle myosin in both groups of MSCs. Histology demonstrated well‐defined clusters of transplanted cells in bladder neck tissues from 4‐56 days after cell injection. The results stress the potential of bone marrow‐derived MSCs to differentiate into smooth and skeletal muscle cells. An exposure to 5‐AZA is not necessary. Survival and integration of MSCs in bladder neck tissue for up to eight weeks emphasizes the use of autologous MSCs for a cell‐based treatment modality of urinary incontinence.

PP 72 Human abdominal and gluteal fat depots: the latter is better!

Adipose tissue is not only the region of energy storage. In addition to the endocrinological role, adipose tissue is also suggested as a reservoir of mesenchymal stem cells (MSC). The goal of this study was to isolate and characterize adult stem/progenitor cells from human gluteal and abdominal adipose tissue. Adipose tissue was obtained from deep subcutaneous abdominal and gluteal fat depots of 10 comparable female patients. Histological evaluation was performed. The isolated cells were investigated by FACS analysis for expression of mesenchymal marker molecules. The proliferation capacity of the cells was analyzed using a BrdU assay. The multipotent differentiation potential into the chondrogenic, osteogenic and adipogenic lineage was assessed by histochemical analysis, immunostaining and RT‐PCR. Adult and juvenile human fibroblasts served as a control.

Histological evaluation did not show an obvious morphological difference. Moreover, no significant differences concerning the proliferation capacity and the expression of cell surface marker molecules were observed between the cell groups. The derived cells achieved the criteria of MSC: they were plastic adherent, expressed the typical CD marker molecules (CD 105, 73, 90) and were negative for CD45 and CD34 and showed a multilineage differentiation potential. Interestingly, we demonstrate that MSC from the gluteal region showed a clear significantly better differentiation capacity into the osteogenic and adipogenic lineage in comparison to cells from the abdominal tissue. Therefore, the gluteal fat depots are likely to deliver MSC of higher potential, which could prospectively be of considerable therapeutic consequence.

PP 73 Application of Epoetin alfa improves renal function during acute ischemic renal failure in an animal model

After ischemic tubular necrosis kidney function can recover. Nevertheless, acute renal failure is an independent risk factor for morbidity and mortality. The goal of this study was to investigate the effect of erythropoietin (EPO) on renal repair mechanisms and renal function in an in vivo rat model of renal ischemia. One hour after 30 min‐ligation of the A. renalis on both sides, EPO (1.000 IE/kg i.p.) was administered to male Wistar rats. A sham‐operated group and non‐operated animals served as controls. Renal function was assessed after 1, 3, 5 and 7 d post application of EPO by measurement of serum biochemical parameters (urea, creatinine) and analysis of functional parameters (perfusion flow rate, glomerular filtration rate, albumin excretion rate, fractional albumin and inulin clearance, 0₂‐consumption rate and fractional sodium‐, water‐, calcium, as well as glucose‐reabsoprtion rate), the latter were obtained using the model system of the isolated perfused kidney. Apoptosis was evaluated by detection of Caspase‐3 expression in the ischemic kidneys. The morphology of acute tubular necrosis was assessed by histological analysis.

Solely after application of EPO we found a population of CD133‐positive progenitor cells in peripheral blood by fluorescence activated cell sorting. Immunhistological analysis confirmed the hypothesis that CD133‐positive progenitor cells contribute to vascular structures in ischemic kidneys. This neovascularisation was enhanced after treatment with EPO. This may be one possible explanation for the functional improvement of ischemic kidneys detected under the influence of EPO in this study.

PP 74 Biological vascularized scaffold: Basis of a liver cell module

We've developed a vascularized liver module, which enables a physiological co‐culture of hepatocytes (HC) and endothelial cells (EC). Basis is a vascularized matrix which provides a blood vessel network for EC an HC co‐culture and the transport of nutrients, metabolites and gases or the removal of toxic metabolites by physiological perfusion. The used vascularized matrix is chemically acellularized and consists of a porcine jejunal segment with a maintained vascular system including an arterial inflow and venous reflux. The vascular structures are reseeded with ECs or progenitor cells (PC) from the bone marrow aspirate. After 7‐14 days hepatocytes are seeded on the matrix lumen to start the co‐culture. During the cultivation period the matrix is perfused with medium over the artery in a bioreactor system. The flow rate is controlled (sensor, computer) to simulate in vivo blood flow. The cells were characterized for the expression of liver specific markers, vitality and for metabolic activities. Human ECs/PCs were seeded successfully via the arterial inflow in the vascular bed of the matrix. Vital human HCs could be repopulated on the surface of the lumen. The culture of HCs on the matrix shows good results for vitality, conservation of liver‐specific functions and formation of cell‐matrix contacts.

Our aim's the development of a vascularized liver module with physiological liver cell functions for pharmaceutical substance screening. Furthermore the system could be enhanced with other cell types (e.g. stem cells) for special applications. A future challenge is the construction of a liver transplant to reduce transplant shortage.

PP 75 Development of a porcine large animal model for an endoscopic therapy of urethral strictures with autologous urothelial cells

To date, no tissue‐engineered based therapy of urethral defects is established. Transplanted autologous urothelial cells promote urothelial regeneration. Thus fibrosis and recurrent strictures might be reduced. We developed a large animal stricture model as pre‐stage for urethral stricture repair with autologous urothelial cells. In male minipigs urethral strictures were placed with ligation and thermocoagulation. For urinary diversion a vesicostomy was done; urothelial cells were isolated from the vesicostomy bladder tissue and expanded in complete keratinocyte serum‐free medium. Four weeks later an internal urethrotomy was performed. In the incised area autologous urothelial cells (PKH26 fluorescent dye labelled and mixed with stabilized hyaluronic acid) were applied with a catheter. Four and 8 weeks, respectively later the animals were sacrificed. The epithelial phenotype of transplanted cells was analysed via immunohistochemistry using pan‐cytokeratin (AE1/AE1). Formation of collagen type I/III was evaluated after picrosirius red staining.

Stricture formation could be detected by urethrography and was histologically confirmed. The stricture areas showed a modified collagen composition, in which collagen type I was 4.5‐fold higher expressed compared to native tissue. Transplanted autologous urothelial cells could be detected in the area of stricture resection. The labelled cells integrated in the damaged urothelium were AE1/AE3 positive. Survival and integration of implanted autologous urothelial cells in the damaged urothelium might support the regeneration of damaged urothelium. The successfully developed stricture model enables investigations on tissue engineering‐based treatment options for urethral stricture therapy.

PP 76 Central necrosis in collections of transplanted cells in muscles of nonhuman primates: implications for cell transplantation

Numerous myogenic cell transplantations implicate the direct injection of cells into the skeletal or cardiac muscles. In this case, the implanted cells form avascular collections. Since the diffusion of oxygen and nutrients from the surrounding tissue throughout the implanted cell collections may be restricted, we hypothesized that ischemic necrosis could develop. We studied this possibility in nonhuman primates. Macaca monkeys were injected intramuscularly with different amounts of myogenic cells per single site. The cell‐transplanted sites were sampled one hour later and at post‐transplantation days 1, 3, 5 and 7, and analyzed by different histological techniques. One day post‐transplantation, the largest pockets of implanted cells showed cores of massive necrosis. The peripheral layer of living cells was roughly 100‐200 micrometers. Delivering 0.1 × 106 and 0.3 × 106 myoblasts did not produce ischemic necrosis; pockets of 1 × 106, 3 × 106, 10 × 106 and 20 × 106 myoblasts exhibited respectively a mean of 7%, 13%, 45% and 65% of central necrosis. An intense macrophage infiltration took place in the muscle, invading the necrotic regions and eliminating them by post‐transplantation days 5 to 7. Therefore, the desire to create more neo‐formed muscle by delivering more cells per injection site is confronted with the fact that the acute survival of the implanted cells is restricted to the thin peripheral layer than can profit of the diffusion of oxygen and nutriments from the surrounding tissue. These observations condition the characteristics of cell transplantation based on direct injection of cells into the tissues.

PP 77 Intramuscular migration capacity of transplanted myoblasts after subcutaneous implantation: a study in nonhuman primates

The main limitation of intramuscular myogenic‐cell transplantation is the fact that the transplanted cells fuse only with the myofibers reached by the injection. This phenomenon is traditionally attributed to a “lack of migration” of the transplanted cells. However, recent observations of the author in monkeys suggest that intramuscularly implanted myoblasts fuse with myofibers following an intra‐fascicular migration, though this migration seems to be only towards the myofibers damaged by the injections. To verify the migration capacity of the implanted myoblasts, we perform an experiment in cynomolgus monkeys. Skeletal muscles of nonhuman primates were damaged by performing 100 parallel needle penetrations per cm² with a 27G needle, 1 hour before injecting ½ ml of a cell suspension of allogeneic β‐galactosidase‐labeled myoblasts subcutaneously under the damaged region and under non‐damaged regions. Monkeys were immunosuppressed with tacrolimus. One month later, the muscle regions under the subcutaneous injections were biopsied and analyzed by histology. In the damaged regions, β‐galactosidase‐positive myofibers were observed up to 1 cm distant from the muscle surface. The distribution pattern of the β‐galactosidase‐positive myofibers followed the pattern of the previous needle trajectories. This indicates that in nonhuman primates the implanted myoblasts are able to migrate for long distances into the recipient's muscle, although to reach and to fuse with damaged myofibers. The problem is thus not a “lack of migration capacity” but rather the incapacity to diffuse throughout a non‐damaged muscle to fuse indiscriminately and spontaneously with non‐regenerating myofibers.

PP 78 Reagents for Defining the Biological Environment of Three‐Dimensional Cell Cultures

A novel system for the configuration of defined biological environments for three‐dimensional cell culture was developed. This system consists of maleimide‐ and thiol‐functionalized hydrophilic polymers, that can be modified with biofactors, such as adhesion molecules, growth factors, and extracellular matrix molecules and that subsequently can be cross‐linked in the presence of cells to form a hydrogel. The chemistry allows uncomplicated setup of three‐dimensional cell culture conditions in straightforward experimental steps. The selection of synthetic or natural polymers, such as polyvinyl alcohol and polyethylene glycol, or serum albumin, which elicit little or no cellular responses, provides an inert background for determining the effects of added specific biofactors. Different cell types, such as primary chondrocytes, mesenchymal stem cells, Madin‐Darby Canine Kidney (MDCK) cells, NIH‐3T3‐cells, and Human Umbilical Vein Endothelial Cells (HUVEC) were successfully cultivated in different gel combinations. Furthermore, the effect of a fibronectin‐derived adhesion peptide on the formation of cellular extensions in HUVEC cells is demonstrated and morphogenetic development of epithelial cell sheets in gels modified with extracellular matrix molecules is shown. Our approach provides convenient toolbox for establishing defined environments for 3‐D cell culture and thus allows to determine cellular responses to extraneous factors as well as to study cell behavior under more controlled and adaptable conditions compared to traditional three‐dimensional culture media. The hydrogel technology thus provides a versatile matrix that can be used to develop extracellular environments for tissue engineering and regenerative applications.

PP 79 The lily‐of‐the‐Valley fragrance receptor ‐ Potential in prostate cancer imaging

Introduction: The detection of lymph node metastases is decisive for the prognosis and therapeutic strategy in patients suffering from prostate cancer. However, the identification of lymph node metastases by magnetic resonance imaging (MRI) is often very difficult and biopsies are necessary.

Materials: To enable differentiation between prostate cancer cells and other tumor cells by gadolinium tetraazacyclododecane tetraacetic acid (DOTA) we coupled it to aminoundecylic aldehyde. Undecylic aldehyde is the inhibitory ligand of burgeonal, which binds to the lily‐of‐the‐valley fragrance receptor (human odorant receptor hOR 17‐4). Until now no activating or inhibitory ligands of odorant receptors have found use in cancer diagnostics. To track the conjugate by confocal laser scanning microscopy it was further coupled to fluorescein isothiocyanate (FITC).

Results: Fluorescence activated cell sorting (FACS) and confocal laser scanning microscopy (CLSM) showed that the FITC‐labelled conjugate predominantly stained prostate cancer cells (PC3, LNCaP) whereas other malignant cells [colon cancer (Colo205), glioma (U373), osteosarcoma (U2OS), cervix carcinoma cells (HeLa), lymphoma cells (Jurkat)] were not markedly stained. No cytotoxic effects were seen even after long time incubation for 48 hours. Magnetic resonance imaging showed that comparable to FACS and CLSM prostate cancer cells could be differentiated from the other cancer cells upon their shorter relaxation times (higher signal intensity) after incubation with Gd‐DOTA‐FITC‐aminoundecylic aldehyde.

Conclusion: The present investigation showed that a fluorescent Gd‐DOTA contrast agent containing undecylic aldehyde can distinguish prostate cancer cells from other tumour cells. Such agents could be used for future magnetic resonance imaging of prostate cancer lymph node metastases.

PP 80 Periodontal regeneration of maxilla alveolar bony defect repaired with BMP‐2 gene engineered autologus bone marrow mesenchymal stem cells

Introduction: The need of periodontal and alveolar bone regeneration was increased in modern clinical dental therapy. Recently, the application of bone morphogenetic proteins to the periodontal defect presented limited regenerative ability of such an approach with the establishment of abnormal periodontal relationship and complications such as ankylosis and root resorption. Short half life of rhBMP‐2 and the need supraphysiological concentration also demonstrated the shortage of protein regeneration therapy. The purpose was to evaluate the regeneration of periodontal apparatus in critical maxilla alveolar bony defects by BMP‐2 gene engineered autologus bone marrow mesenchymal stem cells.

Materials & Methods: Twelve critical maxillary periodontal defects were surgically created over the premolar area combined with root denuded at least 2 × 1 cm2 below cementoenamel junction in 9 matured male miniature swine. Bilateral maxillary defects were randomly assigned into 2 groups: BMP‐2 gene engineered MSCs/polymer (BMP‐2 group), and MSCs/polymer as control. Histological examination was performed at the end of study to exam the microscopic healing and relationships between bone, periodontal ligaments, cementum, and tooth surfaces. Three‐dimension computerized tomography was utilized to calculate the total volume of bone regenerated during experiments.

Results: Our data demonstrated the regeneration of periodontal apparatus was significant better in BMP‐2 group. The 3D CT revealed larger amount of bone formation in BMP‐2 group. New cementum with Sharpey's fiber was observed on the instrumented denuded root surfaces in BMP‐2 group. The control group showed fibrotic healing replacing the bony regeneration. This approach is suitable for periodontal regeneration and may achieve healthy periodontal relationship.

PP 81 Human stem cell sheets engineered on polyelectrolyte multilayer coatings for prospective transplantation

Transplantation of intact, dense cell sheet structures has emerged as promising alternative to cell delivery via injection or biomaterial vehicles. Using of temperature‐responsive polymer coatings has been already successfully employed for non‐enzymatic collection of dense monolayers from different cell types. We sought to develop alternative electro‐sensitive polyelectrolyte multilayer (PEM) coatings for engineering of transplantable cell sheet structures from human multipotent mesenchymal stem cells (hMSCs). As experimental stem cell types we used: adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) and placenta‐derived mesenchymal stem cells (PD‐MSCs). Model PEM coatings of variable thickness (3, 6, and 9 layer pairs) were deposited as alternating layers of positively charged poly‐L‐lysine [PLL] and negatively charged hyaluronic acid [HA]. Further, chemical cross‐linking for substrate stiffening and surface functionalization with fibronectin was explored. Our initial analysis revealed that: (1) both PD‐MSCs as well as AT‐MSCs do not interact well with native, “soft” PEM substrates, (2) stiffness of the [PLL‐HA]n multilayer coating is a critical regulator for adhesion, spreading, and formation of dense sheets from hMSCs, (3) covalent affixation of PEM bound fibronectin is necessary for cell sheet integrity, (4) resulting dense stem cell sheets maintained their vitality and capacity to differentiate into osteogenic lineage. In summary, we established conditions for creation of cell sheet structures from human mesenchymal stem cells (AT‐MSCs and PD‐MSCs) that have a potential for its charge‐mediated collection in intact form for prospective in vivo transplantation, or assembling of in vitro tissue models.

PP 82 Intramyocardial Implantation of CD 133+ Stem Cells Improve Cardiac Function without Bypass Surgery

Introduction: Cell transplantation for myocardial regeneration has shown beneficial effects on cardiac function after myocardial infarction. Most clinical studies of intramyocardial cell transplantation were performed in combination with coronary artery bypass grafting (CABG). The contribution of the implanted stem cells could not be clearly distinguished from the effect of the CABG surgery up to now. Our current phase 1 clinical study has focused safety and feasibility of CD 133+ ‐enriched stem cell transplantation without CABG and the potential beneficial effect on cardiac function.

Method and Results: 10 patients with end‐stage chronic ischemic cardiomyopathy (ejection fraction less than 22%) were selected for the study. Bone marrow (up to 380 ml) was harvested from the iliac crest. CD 133+ cells were purified from bone marrow cells using the CliniMACS® device with purities up to 99%. Autologous bone marrow CD 133+ cells (2‐10 × 106 cells) were injected into predefined regions. Cardiac functions prior to as well as 3, 6 and 9 month after cell transplantation were assessed by cardiac magnetic resonance imaging. Stem cell transplantation typically improved the heart function stage from NYHA/CCS class III‐IV to I–II. The mean preoperative and postoperative ventricular ejection fractions were 15.8 ± 5% and 24.8 ± 5%, respectively.

Conclusion: CD 133+ injection into ischemic myocardium surgery was feasible and safe. Stem cell implantation alone improved cardiac function in all patients. This technique might hold promise as an alternative to medical management in patients with severe ischemic heart failure who are ineligible for conventional revascularization.

PP 83 NGFR: a new osteogenic differentiation marker in mineralizing periosteal cells

Objective: New stem cell sources are required to supply the needs of bone regeneration in Oral and Maxillofacial Surgery. Our study aims at finding differentiating markers which distinguish between mineralizing and non‐mineralizing periosteal‐derived progenitor cells (PDPC) to improve the success of tissue engineering applications. Nerve growth factor receptor (NGFR/CD271) represents a specific marker for bone marrow stem cells. While NGFR expression is not prominent in undifferentiated PDPC, we analysed its expression during osteogenesis.

Methods: Mineralization capacity of PDPC was detected by alizarin and von Kossa stainings. We screened the expression of several monoclonal antibodies in mineralizing versus non‐mineralizing cells by flow cytometry and found the nerve growth factor receptor (NGFR) to be one differentiating marker. NGFR protein expression was additionally visualized by immunohistochemical stainings and semi‐quantitated by western blot. Gene expression analyses followed in both cell groups by quantitative PCR.

Results: We found two PDPC groups which act differently during osteogenesis: the mineralizing cell group was Alizarin‐ and von Kossa‐positive (n = 7), whereas the non‐mineralizing cells showed negative stainings (n = 7). By FACS‐analyses, NGFR was induced during osteogenesis at higher levels in mineralizing PDPC in comparison to non‐mineralizing PDPC. We obtained similar results by immunohistochemical stainings and western blot analyses. Significantly higher transcript levels were detected by quantitative real‐time PCR in mineralizing PDPC in comparison to non‐mineralizing PDPC.

Conclusion: We conclude that NGFR represents a potential differentiating marker which distinguishes between mineralizing and non‐mineralizing periosteal cells during osteogenesis. Its function during the differentiation process should be clarified in further studies.

PP 84 Effect of collagen I on the behaviour of mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are good candidates for bone regeneration because they can be easily obtained and are able to differentiate to osteoblasts. Protein or peptide coatings of bone‐replacing materials can influence survival and in vivo performance of hMSCs. Collagen I, a native bone protein, might be very appropriate for betterment of synthetic implants. The goal of this study was to evaluate the effect of collagen I on hMSC proliferation, adhesion, migration and differentiation. HMSCs were cultured on collagen I‐coated surfaces. This resulted in an enhancement of their proliferation and adhesion. We estimated the amount of formed focal contacts. In hMSCs grown on collagen I, the focal contacts occupied up to 9% of the cell terrain. In order to monitor the behaviour of hMSCs on collagen I, we performed time lapse imaging and we observed that hMSCs were less migratory on collagen I. Next, we induced osteogenic differentiation of hMSCs in vitro. Our results demonstrated: firstly, that collagen I augmented hMSC differentiation and secondly, after we applied blocking antibodies or shRNA against major collagen I‐binding integrins, that the disruption of hMSC‐collagen I interactions led to reduced cell survival and matrix mineralization. Altogether, the contact of hMSCs to collagen I has positive effect on cell proliferation, adhesion and differentiation. Hence, coating with collagen I or related peptides can be implicated in the development of bone‐like biomaterials.

PP 85 Low oxygen tension reduces the osteogenic potential of rat bone marrow mesenchymal stromal progenitor cells

Low oxygen tension may modify mesenchymal stromal progenitor cells (MSC) characteristics and thus influence tissue reparation processes. Such modifications were shown as to MSC proliferation, viability and they are especially important in relation to MSC differentiation potential. There are controversial data on MSC osteogenic differentiation under hypoxia conditions. The aim of this study was to evaluate the impact of reduced oxygen tension on rat bone marrow MSC osteogenic differentiation. MSC at 1‐4 passages were cultivated either in normoxia (95% air + 5% CO₂) or hypoxia and anoxia (95% gas mixture of 95%N2 + 5%CO₂ and 5% or 1% O₂). MSC surface markers expression was analyzed by flow cytometry. Osteogenic differentiation of MSC was determined as to alkaline phosphatase (AP) activity and calcium deposition assessed using Alizarin Red staining. The amount of cells in which both spontaneous and induced AP activity was revealed decreased with the oxygen level reduction. AP activity was significantly reduced but was still observed under anoxia, which indicated the early stages of osteogenic differentiation. MSC viability imposed limits on its further progression with calcium deposition under these conditions. Matrix mineralization was observed only in case of induced differentiation and its extent was less in hypoxia compared to normoxia. These results indicate that low oxygen tension reduces the osteogenic differentiation of MSC and probably help maintain the MSC stemness.

PP 86 Detection of cells with stem cell characteristics in human epidermis

Increased aldehyde‐dehydrogenase (ALDH) activity is a well known marker for hematopoetic stem cells. The ALDH activity can be monitored using the artificial substrate ALDEFLUOR®. Degradation of ALDEFLUOR® by ALDH activity releases a fluorophore, which can be quantified in the cells by flow cytometry. To our knowledge, there is no information about ALDH expression in epidermal stem cells. We analysed the ALDH activity of cells from epidermal cell preparations from human skin biopsies by flow cytometry. Interestingly, in our experiments ALDH positive cell populations do show a costaining with well known surface markers discussed as epidermal stem cell markers integrin beta1 and integrin alpha6. Further physiological analyses may show the stem cell activity of the ALDH/intregrin costained cells.

PP 87 Biodegradable nerve guide conduit with pro‐angiogenic potential

To bridge gaps of lesioned nerves after PNS injury we have started to develop a nerve guide implant made from the special gelatine‐based product NerVusion. Current implants approved for human application do not allow regeneration across gaps of more than a few centimeters in length, possibly due to insufficient blood vessel formation. The novel concept is to design an implant material that facilitates angiogenesis which in turn improves neuronal regeneration. Our tubular implant shall have an inner membrane tube and an outer pro‐angiogenic sponge layer for enhanced blood vessel formation. The NerVusion specimens were produced with defined characteristics with regard to reproducible membrane homogeneity and sponge pore diameter and physicochemical features such as mechanical stability. Preconditioned sponge materials were positively tested in vitro with regard to four parameters: biocompatibility, endothelial cell adhesion, ‐migration and ‐proliferation. Implantation onto the chick egg chorioallantoic membrane induced robust formation of blood vessels. After subcutaneous implantation of sponges into mice, immigration of cells and subsequent formation of functional vasculature became evident. Preconditioned tubes made from Nervusion were biocompatible and were sufficiently permeable to allow outgrowth of neurites and diffusion of doxycycline for reporter gene expression in transfected Neuro‐2a cells in vitro. Intraneuronal implantation in rats displayed no adverse response but long‐term implant stability in vivo.

In summary, the novel matrix based on NerVusion is expected to be a valuable component of future implants in order to improve regeneration by concomitant pro‐angiogenic stimulation.

PP 88 Comparison of adipose‐derived stem cells and bone marrow‐derived stem cells in cell transplantation therapy against brain ischemia

Introduction: Transplantation of bone marrow‐derived or adipose tissues‐derived mesenchymal stem cells (MSC) is now increasingly expected to attenuate the functional deficits after brain ischemia. Here we compared therapeutic potential of these MSC for preservation and recovery from tissue damages under brain ischemia.

Methods and Results: Adipose tissue‐derived stem cells (ADSC) and bone marrow‐derived stem cells (BMSC) were prepared from C57BL/6J mice. Cultured ADSC showed higher proliferation than BMSC. Vascular endothelial growth factor (VEGF) and Angiopoietin1 (ANGPT1) concentration in the cell culture supernatants was measured by ELISA. ADSC demonstrated higher VEGF expression and slightly lower level of ANGPT1 than BMSC. The left middle cerebral artery (MCA) occlusion was made in age‐matched 8weeks C57BL/6J male mice, and the ADSC or BMSC were injected via tail vein just after the occlusion. Assessment of ischemic damages was performed after 90 min occlusion followed by 24 h reperfusion. MSC transplanted group resulted in better recovery than vehicle group. A few cases of hemorrhagic infarction occurred in ADSC group. VEGF is reported to induce strong angiogenesis after brain ischemia, however, it is also indicated to enhance vascular permeability. Increased VEGF can play a role for the hemorrhage and ANGPT1 may contribute to reduce hemorrhagic infarction in the ADSC group.

Conclusion: ADSC exhibit superior efficiency for preserving brain tissue integrity and function after cerebral ischemia than BMSC. Our study also suggest the possibility that modulation of VEGF or ANGPT1 expression in ADSC can be necessary for a safer cell therapy against brain diseases.

PP 89 Efficacy of various durations of in‐vitro predegeneration on the cell count and purity of rat Schwann cell cultures

For reconstruction of peripheral nerve defects, tubulization with artificial grafts coated with cultivated Schwann cells (SC) is a promising approach. By in vitro‐predegeneration of a nerve, efficacy of cell cultivation can be enhanced. This study aimed to improve efficacy of SC‐culture comparing three time spans of predegeneration. Sciatic nerves of 6‐8 week old Lewis rats were harvested and subjected to either 2‐day, 7‐day or 14‐day predgeneration in DMEM, 10%FCS and 1%Pen/Strep. Afterwards, tissue was dissociated in 0.125% collagenase IV and 0.25% trypsine for 24 hours and given into modified melanocyte‐growth medium. Fibroblast growth factor was added at 10ng/mL, in addition to 2μM Forskolin and 5μg/ml bovine pituitary extract. Cell count was determined after dissociation, cell purity was determined 16 hours after cell attachment by means of microscopy. Special value was set on SC to fibroblast relation. Cumulative cell count in culture was 5.8 × 10⁴ for 2 days, 1.12 × 10⁵ for 7 days and 1.48 × 10⁵ for 14 days of predegeneration. Culture purity was approximately equal for 2 and 7 days of predegeneration (88% SCs, 12% fibroblasts after 2 days, 85% SCs, 15% fibroblasts after 7 days). After 14 days of predegeneration, cultures were significantly higher debased by fibroblast proliferation (57% SCs, 43% fibroblasts). The yield of cultivated rat SCs is duplicated by 7‐days in vitro predgeneration in comparison to 2‐days. After 14‐days predegeneration, culture debasement with fibroblast is significantly higher. Therefore, 7‐days in vitro predegeneration of the harvested nerve tissue is an advisable procedural method in the cultivation of rat Schwann cells.

PP 90 Guidance of axons on pre‐aligned Schwann cells on hundreds of polymer filaments for nerve regeneration

The peripheral nervous system is capable to regenerate partly because in many cases only cell processes become lesioned, whereas cells remain viable. Lesioned axons can regenerate if the continuity of the nerve tissue is preserved. Within the nerve, axonal regrowth occurs along thousands of aligned Schwann cells (SCs), so called bands of Buengner. To improve the therapeutic performance of synthetic implants used to bridge nerve gaps we have developed biohybrid bands of Buengner inside nerve guides. 15‐30 μm thick micro‐structured poly‐caprolactone (PCL) and poly‐dioxanone (PDO) filaments were produced by a melt extrusion process. Each filament had six longitudinal micro grooves. To overcome the non‐permissiveness of the polymer filament surface, different plasma treatments and subsequent coatings with a synthetic polyamine and an extracellular matrix protein were investigated. Seeding of pretreated filaments with SCs resulted in longitudinal cell alignment along the micro groove. A metabolic labeling technique revealed that SCs retained their proliferation potential under these conditions. When dorsal root ganglions were explanted onto SC‐seeded filaments, a highly oriented axonal outgrowth could be induced. Implantation of cell‐free‐polymer filaments into lesioned sciatic rat nerves resulted in complete coverage of the implant by invading cells. Currently, implantation of cell‐seeded polymer filaments are under progress. In addition, in order to foster SC immigration we analyzed SCs in vitro and found that recombinant neuregulin‐1 (NRG‐1) accelerated cell migration. Therefore, the future addition of NRG‐1 to nerve guide tubes with polymer filaments might accelerate SC recruitment from both neighboring nerve endings and in turn aid axonal regeneration.

PP 91 Alginate‐based anisotropic capillary hydrogels as scaffolds for guided axon regeneration

Unidirectional diffusion of divalent cations into a sol of sodium alginate created hydrogels with highly anisotropic structures exhibiting capillaries which were circular in cross‐section and parallel to each other. These anisotropic capillary hydrogels were introduced into a dorsal root ganglion (DRG) axon outgrowth assay in vitro to assess their capacity to promote longitudinally directed axon regeneration. Diameter and density of the capillaries were defined by the type of cation used for the gel formation process. Diffusion of barium ions created gels with a capillary diameter of 10 μm and a capillary density of 960/mm², whereas nickel ions generated gels with a capillary diameter of 120 μm and a capillary density of 30/mm². After gel formation, the toxic cations were exchanged by protons without destruction of the capillary structure. The removing of cations was monitored by atomic emission spectrometry. To enhance mechanical stability and decelerate biodegradation, the gels were chemically cross‐linked with an aliphatic diisocyanate. Using the DRG axon outgrowth assay, the stabilized capillary hydrogels elicited highly oriented linear axon re‐growth. It was found that axon ingrowth into capillary hydrogels was influenced by the presence of growth‐supporting cells, i.e. Schwann cells migrating from the DRG into the capillaries. Furthermore it was observed that a higher number of axons and longer axons were generated in gels exhibiting smaller capillary diameters. From these results and further in vivo spinal cord experiments it is obvious, that alginate‐based anisotropic capillary hydrogels represent a promising strategy to induce axon re‐growth following nerve injury.

PP 92 Recapitulating Organ of Corti Development in an in vitro Culture Assay

The mature organ of Corti, the auditory epithelium of the mammalian inner ear, shows a absent intrinsic regenerative capacity resulting in irreversible loss of sensory function after damage to its sensory cells. Due to the limited accessiblity within the temporal bone, it is challenging to apply common imaging and molecular biological techniques to the inner ear.

To study developmental and regenerative issues we established an in vitro model of the inner ear sensory epithelium. Isolation of murine stem cells from the postnatal organ of Corti (postnatal day 5) allowed us to grow proliferating spheres under suspension culture conditions shown by labelling with proliferation markers PCNA, Ki67 and BrdU. Transfering these spheres to adherent culture conditions resulted in monolayered epithelial patches, that exhibit characteristics of the otic sensory epithelium. Typical hair cell markers (Math1, Myosin VI and ‐VIIA) and supporting cell markers (p27Kip1, S100 and Prox1) were detected in these patches.

Reexpression of these proliferation and differentiation markers indicates recapitulation of embryonic development of the organ of Corti in our in vitro model. This model opens the avenue to analyse mRNA‐ and protein expression with quantitative methods, for example by flow cytometry. Supported by: BMBF 0313612A

PP 93 Transretinal electrical stimulation induces neuroprotective effects in a mouse model of retinitis pigmentosa

Electrical stimulation has been shown to have neuroprotective effects on ganglion cells and photoreceptors in axotomized and dystrophic retinas from Royal College of Surgeon (RCS) rats. This study determined whether electrical stimulation also has a neuroprotective effect on cells in the inner nuclear layer (INL) of retinas. We cultivated retinas from adult RCS rats on microelectrode arrays and stimulated them continuously with 20 Hz for up to six days. Afterwards, we subjected them to quantitative immunohistochemical analysis. Using TUNEL assay and antibody staining (OX42, ED1) we found that transretinal electrical stimulation (TRES) with charge densities within the range of 100 to 500 μC/cm² resulted in a lower number of apoptotic neurons in the INL of degenerated retinas from RCS −/− rats after one day of continuous stimulation and reduced activation of migroglial cells in RCS −/− and congenic control (RCS+/+) rat retinas. The effect of electrical stimulation on apoptosis and reduced activation of migroglial cells was closely correlated with the strength and duration of the stimulation. The neuroprotective effect of TRES on neuronal cells in the INL of degenerated RCS rat retinas supports the idea that electrical stimulation may be a therapeutic option to delay the progression of retinal degeneration in patients suffering from retinitis pigmentosa.

PP 94 Mesenchymal stem cells gain multiple neuroprotective properties upon pro‐neural conversion

Specific culture conditions (suspension and serum‐free culture at low oxygen) for adult human mesenchymal stem cells (MSC) lead to epigenetic modifications of gene expression (here referred to as ‘conversion’). Neural genes as well as many neurotrophic factors are up‐regulated. Compared to genuine human neural tissue the expression of neural genes (e.g. tubulin beta3, glial fibrillary acidic protein) remains at low level, but the expression of neurotrophic factors, i.a. hepatocyte growth factor and vascular endothelial growth factor, obtains levels of functional importance on the cellular level. Culture of rodent neural stem cells (NSC) with conditioned medium from converted MSC and direct co‐culture experiments reveal distinct effects on the proliferation rate, cell survival, and differentiation potential of NSC. Additionally, converted MSC significantly protect NSC against the neurotoxin 6‐hydroxydopamine. Genome wide gene expression analysis, real‐time RT‐PCR, immunohistochemistry, and flow cytometry reveal a very high up‐regulation of F‐spondin and neprilysin upon pro‐neural conversion, two genes which are known to reduce the production or increase the degradation, respectively, of the Alzheimer's disease related amyloid beta peptide. In summary, though we do not achieve neural transdifferentiation of MSC by using our protocol, we show that MSC gain various neuroprotective properties upon pro‐neural conversion.

PP 95 Carrier matrix for siRNA‐mediated gene knock down to promote neuronal regeneration

Axons in the adult vertebrate central nervous system (CNS) exhibit almost no regeneration after injury, partly because of the presence of neurite outgrowth inhibitors in the CNS. The aim of this study is to enhance functional recovery and axon regrowth using siRNA in order to transiently inhibit the intracellular signalling pathway of central components into which various repulsive inputs converge. An effort concerning this objective is to find an appropriate carrier matrix for binding, stabilization, and promoting cellular uptake of siRNA.

Chitosan is a biocompatible and biodegradable material which enables a strong electrostatic binding of oligonucleotides. In this study we present the effective binding of siRNA to chitosan under formation of nanoparticles. Analysis of different kinds of chitosan‐siRNA nanoparticles using gel‐retardation assays demonstrated high stability of those complexes abrogated only after addition of competing agents. Finally, cell transfection experiments with fluorescently labelled siRNA showed efficient uptake of chitosan‐siRNA nanoparticles into different cell types which play an important role in the nervous system.

Our results therefore highlight the potential application of chitosan in siRNA‐mediated gene knock down to promote neuronal regeneration after spinal cord injury.

PP 96 Neural stem and progenitor cells from the human adult gut: A novel cell source for the treatment of neurointestinal disorders

Neural stem and progenitor cells from the enteric nervous system have been postulated as an appropriate cell pool for future cell‐based therapies against specific neurogastrointestinal disorders such as hypo‐ or aganglionosis. Recently, the generation of enteric neural progenitors from human neonatal and early postnatal gut (up to 5 years after birth) was demonstrated. In the present study, we describe the successful proliferation and differentiation of enteric nervous system progenitors isolated from human adult gut. We were able to generate proliferating neurosphere‐like bodies from adult small or large human gut tissues, which could be sub‐cultivated and maintained over several weeks in vitro, depending on the applied cell culture conditions. Spheroid‐derived cells were differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system as demonstrated by in situ hybridization, immunocytochemistry, RT‐PCR and electrophysiological studies. Implantation experiments into intestinal organotypical slice cultures and aganglionic gut segments of fetal mice showed the integration and differentiation capacity of human progenitors. Our results demonstrate the feasibility of isolating and expanding functional enteric progenitors. These findings offer new strategies for enteric stem cell research and cell‐based therapies.

PP 97 Multipotent stem/Progenitor cells and Their derivatives promote adult rat DRG neurite outgrowth

In an attempt to support axon regeneration across CNS or PNS lesions, we are currently investigating the in vitro growth promoting properties of different cell types. Therefore, highly enriched populations of human mesenchymal stromal cells (hMSCs), normal human neural progenitors (NHNPs), immature type I astrocytes (itIACs) derived from the NHNPs as well as primary rat astrocytes (prACs) have been investigated in simple 2D cultures using freshly dissociated adult rat dorsal root ganglion (DRG) neurons.

Confluent cultures of hMSCs grow in an orientated manner, promoting the regeneration of large numbers of primary neurites from the dissociated DRG, significantly exceeding the numbers of primary neurites counted on the other investigated cell types. Neurites of dissociated DRG neurons rapidly extend highly directed neurites which follow the orientation of the long axis of the densely packed hMSCs, suggesting that these cells may not only support axon regeneration but may also influence its orientation. A random, non‐oriented outgrowth of neurites was observed from DRG plated onto PLL/laminin‐coated surfaces. Despite the hMSC‐induced induction of large numbers of neurites and the orientated tendency for neurite outgrowth, maximal overall neurite length, longest individual processes and outgrowth per neurite branches were supported best by the immature human type I astrocytes.

Therefore, the axon growth promoting property of stem/progenitor‐derived immature human type I astrocytes was found to be superior to the other entire cell types tested, suggesting that such cells may be of interest in promoting tissue repair and functional axonal regeneration in models of spinal cord injury.

PP 98 Expression of pluripotency genes in cells derived from the SVZ or olfactory bulb of the adult mammalian brain

The subventricular zone (SVZ) of the adult mammalian brain harbours Nestin/GFAP double positive cells that function as neural stem cells (adNSC). These cells divide to generate transit amplifying cells (adPCi), which in turn give rise to progenitors (adPCl) that migrate in the rostral migratory stream to their final destination in the olfactory bulb (olfB) where they terminally differentiate. We investigated co‐expression of pluripotency genes in SVZ and olfB‐derived primary cell lines, since their expression might be critical for self‐renewal and maintenance of the stem cell‐ and progenitor‐state. SVZ‐ and olfactory bulb‐derived cultures contained Neurofilament+, Nestin+ and Nestin+/GFAP+ cells, as well as few other cell types. This was similarly observed for explants from mouse, rat and human brain. After the first round of amplifiaction the cultures consisted mainly of Nestin+/GFAP+ cells. In general, the proliferation rate of early primary cultures was high. Rodent, but not human Nestin+/GFAP+ cells amplified well in culture. In all cases, cells grew in a matrix‐dependent fashion as monolayer or neurosphere cultures. E.g, rodent Nestin/GFAP double positive cells formed (i) monolayers on standard Polyornithin‐ and Matrigel‐ but not on Laminin‐matrices, and (ii) increasing concentrations of fibronectin, permitted adherent growth of all rodent and human cell lines. The stemness genes oct4 and sox2 are co‐expressed in all rodent cultures but down‐regulated with increasing passages, irrespective of their high proliferation rate. Nearly invariant expression of nanog was observed in the human cells, whereas the expression level of both oct4 and sox2 varied largely amongst the various human cell lines.

PP 99 Expression of stemness genes in neural stem cell‐like cells derived from malignant glioma

The survival rate of patients with malignant brain tumors is very poor. It is not known whether and how cells with stem cell features affect the growth of glioma or the success of chemotherapy and irradiation. We evaluated the presence of stem cell‐like cells in different types of malignant glioma and analyzed the expression of the stemness genes oct4, sox2 and nanog. Primary cultures of glioblastoma and gliosarcoma consisted of varying amounts of fibroblasts, myofibroblasts, astrocytes, neurons and other cell types. A large portion of the latter ones co‐expressed Nestin and GFAP and a varying and usually smaller portion was Nestin+, indicating the presence of NSC‐like cells in glioma‐derived primary cultures. When continuously grown in media containing FCS Nestin+/GAPF+ cells disappeared from the cultures. In contrast, growth in medium containing a serum substitute and mitogens, a condition used for the propagation of adult neural stem cells, led to progressive enrichment of Nestin+/GFAP+ cells, which self‐renewed and formed neurospheres. The proliferation rate of the NSC‐like cells, however, varied largely amongst the various primary cultures and was low for most tumor explants. Nanog RNA was nearly invariantly expressed in both serum‐deprived and un‐deprived cultures. In contrast oct4‐expression (RNA and protein level) and Sox2‐levels (protein) varied significantly amongst the different tumor cell lines, were stronger in serum‐deprived cultures and appeared highest in cell lines derived from two clinically exceptional tumors. Together this indicates that the stemness genes oct4 and sox2 might take a role in maintenance and self‐renewal of NSC‐like glioma‐derived cells.

PP 100 Neural‐Related marker expression of undifferentiated human mesenchymal stromal cells exhibits inter‐Donor variability

In the last few years several studies have reported that bone marrow‐derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries that were thought to be uncrossable. These reports evaluated the existence of neural‐related proteins mostly only after induction of the MSCs. To assess the amount of neural‐related transcripts and to determine the degree of homogeneity between donor samples, we investigated the basal expression of a range of neural‐related mRNAs of non‐differentiated MSCs obtained from 4 healthy donors. This expression analysis revealed that several of the commonly used marker genes were already expressed by undifferentiated human MSCs (e.g. nestin, Enolase2 and microtubule associated protein 1b). Furthermore, mRNA for some of the neural related transcription factors, like Engrailed‐1 and Nurr1 were also strongly detected. However, several other neural‐related mRNAs (e.g. dopamine receptor D2, Enolase2, neurofilament light and myelin basic protein) that could be identified were not expressed in all donor samples. Similarly, synaptic vesicle‐related mRNA, syntaxin could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. These results demonstrate the existence of an inter‐donor variability within human MSC samples that has not previously been described. This donor‐related heterogeneity might affect reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs.

PP 101 Differentiation of adult stem cells towards microglial cells

Introduction: During aging, the role of microglia in the brain changes from ‘friend to foe’ – in young organisms microglia are actively participating in the clearing of protein debris from the brain. Microglia are replenished by adult stem cells (ASC) that migrate into the brain and by proliferation of local cells. In aged and old organisms however, microglial replenishment is impaired. The brain is burdened with ‘old’ microglia that remain in chronic activation and thus cause inflammation and damage in the brain. We therefore wanted to study to in vitro differentiation of adult stem cells from the bone marrow towards microglial cells.

Methods: Adult stem cells were in vitro differentiated using media, including astrocyte conditioned medium, Flt3, TGF‐b, FGF and other substances. Cells were then tested for their surface antigen profile using flow cytometry (CD11b, F4/80, CD45, CD45/B220). Phagocytotic activity and oxidative burst were tested using phorbol ester phorbol 12‐myristate 13‐acetate (1μM PMA) activation. Incorporation, survival and migration of the in vitro derived microglia‐like cells was tested in organotypic brain slices.

Conclusion: We found changes in the expression profile of the cells, showing increased expression of macrophage/microglial marker and haematopoietic markers, including CD45, F4/80 and CD45/B220. In vitro differentiated microglia‐like cells show phagocytotic activity and oxidative burst after stimulation with PMA. Invasion into the brain slice was observed starting at around day 3 and showed only very few apoptotic cells.

PP 102 Scaffold‐Based skin equivalents: In vitro ‐models to study the effects of mature dermal organization on tissue regeneration

Advanced in vitro‐generated skin models gain importance as tools in basic research as well as clinical application in regenerative medicine. Here, we present further details of an improved long‐term skin equivalent (SE) enabling studies on skin reconstruction including maintenance of epidermal stem cells and organisation of incorporated endothelial cells exhibiting tubulogenesis. Keratinocytes were grown on dermal equivalents reinforced by scaffold fibres and colonised with fibroblasts. These scaffold‐based SEs demonstrated sustained epidermal viability over 15 weeks with regular differentiation. The continuous expression of major junctional components indicated the achievement of epidermal stability and barrier function. A progressive maturation of the dermal part of the SEs became evident by a significant remodelling of the extracellular matrix (ECM) changing from an initial provisional status to an ECM with an in vivo‐like composition (containing collagen and elastic fibres). Different from the conventional collagen‐based SEs, this authentic matrix mimics native dermis and provides the microenvironment permitting longevity of label‐retaining epidermal stem cells. Furthermore, it represents a microenvironment ideal for endothelial cells, allowing their self‐organisation into numerous tube‐like structures with endothelial basement membranes confirming the endothelial potential for capillary‐like self‐assembly.

In summary, the long‐term SE presented herein, exhibits a superior epidermal architecture with renormalised differentiation including tissue homeostasis. Its authentic ECM has proven to be a crucial component for successful in vitro vasculogenesis as well as the establishment of a functional epidermal stem cell niche.

PP 103 Ex vivo expanded hematopoietic progenitor cells improve dermal wound healing by paracrine mechanisms

Background: Although dermal wounds are common, treatment remains limited and largely ineffective. Recent studies suggest that therapeutic application of progenitor cells are useful for tissue regeneration.

Objective: We here investigated the effects exerted by the recently characterized immortalized hematopoietic progenitor cell line DKmix and their conditioned medium in a murine wound healing model.

Methods and Results: Injection of both DKmix cells and their conditioned medium accelerated wound repair between day 3 to day 10 compared to PBS‐injected control mice (n = 8, p < 0.01 DKmix cells vs. control, p < 0.01 conditioned medium vs. control at day 6). The treated groups exhibited more CD31+−capillaries at day 6 after injury compared to the control group (n = 4, p < 0.01 DKmix cells vs. control, p < 0.001 conditioned medium vs. control) whereas there was no change in infiltrated CD68+macrophages. Conditioned medium of DKmix cells induced tube formation of human endothelial cells in Matrigel assays (n = 4‐6, p < 0.05 conditioned medium vs. control) as well as migration (n = 4, p < 0.01 conditioned medium vs. control) and proliferation of murine 3T3 fibroblasts (n = 5, p < 0.05 conditioned medium vs. control). Abundant levels of matrix metalloproteinase‐2 and − 9 in the supernatants were detected. Protein arrays of the supernatants revealed a strong secretion of cytokines and growth factors such as MCP‐1 and GM‐CSF from DKmix cells.

Conclusion: DKmix cells improve skin‐substitute wound healing by promoting angiogenesis as well as migration and proliferation of fibroblasts. These data suggest that immortalized hematopoietic progenitor cells significantly improve dermal wound healing by paracrine effects.

PP 104 Identification of Melanoma Cells with Stem Cell Properties

Cancers contain a small population of cells named “Cancer Stem Cells” (“CSCs”) that are believed to play a critical role in the development and progression of the disease. These cells have been found in several types of cancer including just recently melanoma although their characterization is still poorly known. It is also thought that CSCs drive the metastatic spread of cancer. CSCs show resistance to a number of conventional therapies and it is believed that this explains why it is difficult to completely eradicate the disease and why recurrence is an ever‐present threat. We report the isolation and the characterization of a subset of melanoma cells with stem cells properties by two different experimental approaches. The first one allowed us to isolate spheroids by using a stem cell permissive serum‐free medium. The second one allowed us to isolate a side population (SP) based on Hoechst dye exclusion. Interestingly, by comparison to the corresponding parental cell line, we particularly show that the isolated spheroids are (i) not pigmented, undifferentiated; (ii) able to get differentiated in presence of serum; (iii) in vitro more clonogenic and more chemo‐resistant; (iv) display an increased expression of certain ABC transporters, known to be involved in the multidrug resistance (MDR), as well as some specific stem cell molecular markers. Besides, the SP isolated on the basis of Hoechst dye exclusion assay, share many of the above described cancer stem cell characteristics. Selectively targeting of CSCs should definitively offer a new paradigm in both cancer therapeutics and diagnostics.

PP 105 In human fibroblasts CC‐TT transitions of p53 decrease during in vitro proliferation

The proliferation of human skin cells in vitro plays an important role in therapeutic approaches in applied biomedical sciences such as tissue engineering and biomedical analytics e.g. biocompatibility and screening of active agents. But in vitro proliferation of cells is still an artificial system compared to the in vivo situation for example due to higher proliferation rates. For autologous cell transplantation it might be important to know whether potentially existing DNA‐damages increase or decrease during in vitro proliferation. These damages could serve as a quality control marker for skin cells cultured for use in tissue engineering. A well‐known UVB‐induced mutation is the CC‐TT transition in the tumor suppressor gene p53 at codon 281/282. We established a real‐time‐PCR method using SYBR Green technology to detect and quantify these p53 mutations in human dermis ex vivo and during in vitro proliferation of human dermal fibroblasts. Therefore human skin samples were collected and isolation of fibroblasts was performed from the dermis fraction. Later DNA‐extraction was performed every new passage. To represent the situation ex vivo, DNA was extracted from pieces of the dermis. There is a decrease of p53 mutations during in vitro proliferation. These results indicate that there is no accumulation of p53 mutations during in vitro proliferation, so it might be better to extend in vitro proliferation of cells regarding autologous cell transplantation. In contrast there is a hint of an increase of p53 mutations from the ex vivo to the in vitro situation of dermal fibroblasts.

PP 106 Quartz Crystal Microbalance with sensitive surface coatings for perioperative bleeding monitoring

Purpose of the study: In addition to many diseases some extracorporeal operations (open heart surgery, dialysis) bear high risks of bleeding and thromboembolic complications. In such situations time plays a critical role. Therefore it would be an advantage to get realtime access to the major hemostatic data, namely clotting time (prothrombin time), heparin effects, platelet function and fibrinolytic activity. The novel technique of a Quartz Crystal Microbalance (QCM) allows simultaneous measurements of these different haemostatic tests out of citrated whole blood and will give sufficient information about complex bleeding complications.

Methods: The QCM method permits the detection of any adsorbed masses (cells, proteins, particles, etc.) or changes in viscosity in real‐time by changes in resonance frequency. With adequate sensor coatings and reagents, prothrombin time, platelet aggregation and fibrinolytic activity were measured. The measurements were confirmed by comparison with commercially available coagulometers and aggregometers as well as scanning electron microscopy and fluorescence microscopy.

Results: Coagulation times and fibrinolysis were measured in undiluted and diluted whole blood on polyethylene and fibrinogen surfaces. Platelet aggregation was carried out in platelet rich plasma on polysterene and fibrinogen surfaces. The results from these measurements showed good accordance to established methods.

Conclusion and outlook: The innovative QCM method seems to be suitable for real‐time investigation of hemostatic processes. Furthermore we gain to automate the measuring process to enable the attending physician to start an adequate therapy as fast as possible.

PP 107 Matrix elasticity influences the maintenance of the chondrogenic phenotype

Cells sense and respond to the mechanical properties of their environment. It has been demonstrated that matrix elasticity plays an important role not only for cell spreading and adhesion but also for cell differentiation. The aim of this study was to examine the influence of mechanical properties on the differentiation of chondrocytes cultured in monolayer. We used a 2D‐culturing‐system in which polyacrylamide gels with different percentages of BIS‐Acrylamide were coated with collagen type I. This system allows altering the matrix elasticity while leaving all other matrix‐parameters constant.

Matrices with a Young's Modulus of 5kPa, 10kPa, 40kPa and 100kPa were produced, as determined by atomic force microscopy. Porcine chondrocytes were cultivated on matrices in low density culture for up to two weeks. The differentiation of chondrocytes was monitored by measuring collagen type I and II expression. Additionally proliferation and cytoskeleton organisation was investigated.

Obvious differences between cells cultivated on the different matrices were monitored. Porcine chondrocytes grown on stiffer matrices showed a more flattened morphology with highly organised stress fibers, lower collagen type II and higher collagen type I expression when compared to cells grown on softer matrices.

Our findings indicate that chondrocytes sense the elasticity of the matrix. Maintenance of the chondrogenic phenotype is prolonged on soft substrates while proliferation is lowered on these substrates. These data might be used in the design of novel scaffolds with mechanical properties specifically tailored to guide chondrocyte differentiation in tissue engineering applications.

PP 108 Ink‐Jet printing of proteins and functional nanoparticles for automated functionalization of surfaces

Ink‐jet printing is a relatively straightforward fabrication process. The targeted material deposition avoids material spill‐over and is therefore very much applicable for processing of high‐cost materials such as biological substances. Our objective is to identify biocompatible fluids that meet the needs for a stable piezo ink‐jet printing process. We will present results concerning substrate‐activation, ink‐preparation, and the maintenance of biological functions during the ink‐jet process. Recently, ink‐jet printing technology has been used to fabricate electronic, medical, optical and polymeric devices [1, 2, 3, 4]. In contrast to classical coating techniques as spin‐coating, dip‐coating or spray‐coating printing allows for patterned material deposition. Many formats of 2‐D drawings can be rasterized into X‐ and Y‐coordinates and directly be converted into multi‐material patterns via the printing process. The physico‐chemical properties of inks determine their jettability: surface tension and viscosity are two primary chemical properties that determine printing success. The optimum viscosities for jettable fluids in piezo drop‐on‐demand printheads are 5 mPa s to 12 mPa s, surface tension has to be in the range of 24 mN/m to 33 mN/m. However, most biological materials are water‐based and exhibit very low viscosities (0.1 mPa s to 1 mPa s) and very high surface tension values (58 mN/m to 60 mN/m). In order to enable economical biofunctional substrate‐coating and to integrate substrate‐functionalization in a process capable of being fully automated we develop ink formulations containing biomolecules or (bio‐)functional components.

PP 109 Retroviral gene transfer to cells from amniotic membrane of normal embryos of Golden Retriever Muscular Dystrophy Dogs (GRMD)

Embryonic stem cells have become a promising tool to be used in cell therapy for many diseases, especially genetic ones. Duchenne Muscular Dystrophy is a progressive genetic disease that seems to be one candidate to cell therapy with embryonic stem cells. Our group has the best model to study that, a Golden Retriever Muscular Dystrophy dog, which shows the same pattern of the human disease. However, one challenge of cell transplantation is investigate the engraftment of stem cells in the injured site and prove that delivered cells were transformed in local cell lineage. For that, genetic markers should be used. Here, we describe cells derived from Amniotic Membranes (AM) of dog's embryos successfully transducted in vitro by pLNPoZ retrovirus, a Moloney Murine Leukemia Virus vector carrying the reporter bacterial gene LacZ. Retroviruses were produced by PT67 packaging cell lineage and used to infect AM cells. AM cells transduction was confirmed using X‐Gal staining method. Additionally, these cells did not show distinct phenotype in cell culture after virus transduction. Next step, these cells will be transplanted in order to study their comportment and therapeutic effectiveness in vivo.

PP 110 Molecular Imaging: Influence of SPIO‐Labeling on the Functionality of Human Adult Mesenchymal Stem Cells

Purpose: Aim of the study was to evaluate the influence of labeling procedures using clinically approved small particles of iron oxide (SPIO) with or without transfection reagents (TA) on functional parameters of human mesenchymal stem cells (MSC).

Materials and Methods: Adult MSC were isolated from the bone marrow of 7 patients. The MSC populations, characterized by flow cytometry and in vitro differentiation, were labeled with 200 μg/ml SPIO or with 60 μg/ml SPIO alone or in combination with various TA. Directly after labeling and 2 passages after labeling migration assays, quantification of colony forming units and evaluation of the differentiation potential were performed. Quantification of the cellular total iron load, determination of the cellular viability and electron microscopy were performed additionally.

Results: Labeling of MSC with SPIO with or without TA was feasible without affecting cell viability. SPIO in combination with TA coated the cellular surface directly after labeling, but had been incorporated into the cells after 2 passages in culture. Labeling of MSC with TA led to a significant decrease of migration capacity. This effect was abolished after 2 passages in culture. Labeling with and without TA led to a significant decrease of ability of colony formation. This effect could be observed also after 2 passages. Labeling did not affect the differentiation capacity of MSC in vitro.

Conclusion: SPIO labeling with and without TA does have functional effects on human MSC decreasing the migration capacity and the ability of colony formation of the cells.

PP 111 MR‐Visualization of Endothelial Progenitor Cells after labelling with SPIO in a dynamic Cell Culture System

Purpose: The aim of the study was to optimize parameters for labelling of endothelial progenitor cells by incubation with SPIO and visualization of their adhesion in a dynamic cell culture model using explanted porcine vessels.

Material and Methods: EPC were collected from human blood by means of leukapheresis followed by ficoll‐density gradient separation and isolation using CD105 magnetic beads. Cells were incubated with 200 μg/ml SPIO or with 60μg/ml SPIO in combination with transfection agents for 4h and 18h. Vitality was assessed according to the ECE‐method. To determine the extent of the labelling process, photometric iron measurements and static phantom MRI were conducted. In‐vitro adhesion of labelled EPC was imaged using denuded porcine carotid arteries placed into a dynamic cell culture model adapted to small animal MRI Systems at 7T. After imaging, samples were prepared for comparative histologic examination. Next to standard staining, in situ hybridization using a human specific probe to determine the origin of the adhesive cells and prussian blue staining to identify iron were conducted.

Results: Labelling of EPC did not influence vitality of the cells. MRI was able to detect the cells that adhered to the denuded vessels under the dynamic conditions. The imaging correlated with the results of the stained samples gathered from the scanned sections.

Conclusion: Labelling of human endothelial progenitor cells is possible without impairment of cell function and visualization can be conducted in a dynamic cell culture model. MRI was able to depict cell adhesion comparable to histologic staining.

PP 112 Development of a novel phospholipid‐based biomimetic implant surface modification for orthopedic applications

Titanium is known to be a particularly biocompatible implant material. However in some cases subsequent development of interfacial fibrous tissue leading to the isolation of the implants is observed. Phospholipid coatings on titanium implant surfaces have been intensively studied and proved to be a positive factor for cell‐implant interactions in vitro (Willumeit et al., 2007, J. Mater. Sci: mater. Med. 18:367‐380). However, the so far used adsorptive manner of the lipid transfer is not stable enough. The aim of this study was to develop a novel, covalent coating of titanium with a phospholipid biomembrane model and to investigate the behaviour of human bone derived cells (HBDC) on this modified metal surface. Octadecylphosphonic acid (OPA) was first used to form a covalent linked monolayer on surfaces of smooth titanium samples. A phospholipid (POPE) monolayer was then deposited on this support by means of Langmuir Blodgett Technique to achieve a bilayer serving as cell membrane mimic. The quality of the coating was analysed by contact angle, reflectometry and X‐ray photoelectron spectroscopy measurements. For the study of osteointegration cell adhesion, viability and differentiation of HBDC were determined. Scanning electron microscopy was employed to corroborate the results. The data show that a stable biomembrane‐like coating was created. It was observed that this coating enhances cell adhesion and proliferation and allows the cells to produce proteins fundamental for bone growth. Our results suggest the possible usefulness of stable phospholipid based metal coating for implantology.

PP 113 A specific semi‐manual device for rapid repetitive intramuscular cell implantation

Intramuscular transplantation of myogenic cells in nonhuman primates and humans requires meticulous repetitive injections. Performed with syringes operated manually throughout large regions, the procedure takes a lot of time, becoming tiring and imprecise. In order to accelerate this task and to render it more precise, we developed a semi‐manual cell injector. Diverse mechanical principles were examined to choice an optimal arrangement. Simulations were done to verify the concept before machining a functional prototype. The final prototype was able to repeatedly inject a cell suspension simultaneously through six needles or less, delivering few microlitres equally through the intramuscular trajectory of each needle. The device was tested in vivo by performing intramuscular allotransplantations of beta‐galactosidase‐labeled myoblasts in cynomolgus monkeys. The success of the graft was comparable to that of our standard methods but the procedure was 4 to 6‐fold faster. The transplantation is facilitated by several facts.

PP 114 Raman spectroscopy as a noninvasive tool for cellular characterization

Background: The production of autologous transplants on the basis of primary human material is well established and is being applied since several years for example in case of cartilage transplants. But still it is not easy to analyse cell vitality and viability in vivo because most analytical methods fail for monitoring cellular activity in its natural environment without reagents and without destruction of the cells to be analysed. The aim of our research is to deploy a system and method to analyse and monitor the environment, for example contaminations in isolates of human tissue and cells for the production of transplants, and for the monitoring of cell viability and vitality in the production process with Raman spectroscopy.

Methods: Raman spectra were obtained with a device equipped with a 785 nm Laser and an optimized charge‐coupled device (CCD) for NIR wavelengths. A second device (NTegra‐Spectra, NT‐MDT) was used for testing and comparing the measurement results. References for cells were gained at different measuring points inside single cells. The data analysis was done with multivariate methods.

Results: Spectral information and results of the multivariate analysis show that it is possible to detect and analyse different cell types without any additional reagents used and without destroying the cells. The measurements can even be done up to several 100 μm inside transplant materials.

Outlook: As well as the automatization the biological aspect of differentiation and proliferation states of primary chondrocytes will be further investigated to get a valid model for spectral analysis.

PP 115 Design of a GMP conform, disposable bioreactor for mechanically promoted engineering of cartilage

Introduction: Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The application of mechanical stimulation in dynamic bioreactors is considered to be an effective strategy to improve cartilage quality in tissue engineering.

Materials & methods: In this study a novel bioreactor enabling the application of dynamic compression was tested using porcine chondrocytes, in monolayer culture dedifferentiated chondrocytes and mesenchymal stem cells (MSC) in collagen hydrogel. The mRNA and protein levels of two main markers of chondrogenic differentiation (collagen type II and aggrecan) were determined using immunohistochemistry and RT‐PCR. Furthermore the expression of marker genes for chondrogenic dedifferentiation and hypertrophy (collagen type I and collagen type X) were determined.

Results & perspectives: In our studies improved cultivation parameters and dynamic loading conditions provided by a new bioreactor system have been proved beneficial in regard to the cartilage quality and the differentiation status of chondrocytes cultivated in collagen gels in vitro. Furthermore, redifferentiation of dedifferentiated chondrocytes could be detected. Further improved studies will determine the impact of the cultivation in this newly designed bioreactor system on the chondrogenic differentiation of mesenchymal stem cells under in vitro conditions.

PP 116 Cell‐based improvement of neural prosthetic device

Deep brain stimulation in selected brain regions has provided remarkable therapeutic benefits for otherwise treatment‐resistant movement and affective disorders such as chronic pain, Parkinson's disease, tremor and dystonia. Recently, there are several types of neural prosthetic implants in medical use, e.g. to treat Parkinson's disease. Beside psychological side effects several responses of the brain are described in animal studies after implantation of rigid and flexible devices. For long time application the pronounced disadvantage means an encapsulation of the neural prosthetic device by astrocytes and microglia and often leads to an insufficient signalling at the brain‐device‐interface. In this study we tried to circumvent these effects by covering the surfaces of neural prosthetic devices with adult stem cells, which serve as native interfaces. We here analysed the benefit of rat pancreatic and salivary gland stem cells to improve biological and functional integration in the brain exploring an in vivo rat model. Given the fact, that these cells show long time stability and a high proliferation activity they present an alternative source of pluripotent stem cells for various applications. In vitro, we test their growth characteristics on selected flexible prosthetic surfaces and analyse the differentiation ability into neural cells. Cell‐covered electrodes will provide new possibilities for stable chronic integration of flexible multichannel implants in the brain.

PP 117 Amino‐ and carboxyfunctionalized nano‐ and microstructured surfaces for evaluating adhesion, proliferation and differentiation of primary keratinocytes

Background: Tissue Engineering is an interdisciplinary research field with the goal to manufacture in vitro tissues and organs. A crucial factor is the functional cultivation of primary cells. Therefore, scaffolds have to be created, featuring structurally and functionally tailored substrates. To gain basic insight into the impact of non‐biological features on cells' behaviour, keratinocytes were cultured on functionalized planar, nano‐ and microstructured surfaces. Adhesion, proliferation and differentiation of primary keratinocytes have been evaluated.

Methods: Glass‐slides were coated with nano‐ or microparticles and sintered at 560°C for 60 min or 620°C for 90 min. After sintering, glass‐slides were functionalized with Aminopropyltriethoxysilan or Carboxysilantriol. The surface was analyzed with ellipsometry, SEM, XPS and AFM. Cells were isolated from human foreskin and cultured at 37°C and 5%CO2. Adhesion, proliferation and differentiation of primary keratinocytes on various surfaces were evaluated with cell proliferation tests and immunohistochemical staining of cytoskeleton and differentiation markers.

Results: Stable nano‐ and microstructured surfaces were prepared and amino‐ and carboxy‐functionalized. Primary keratinocytes showed better adhesion and proliferation on amino‐ than on carboxy‐functionalized surfaces. Increasing proliferation from microstructured to planar surfaces with amino‐functionalization could be detected by WST‐1 tests. Cytokeratin 14 as an early differentiation marker was detected, especially on modified surfaces, but no later markers for differentiation like cytokeratin 10 or filaggrin were found.

Outlook: Insight into the cells' preferences concerning structure and chemical functionality of the substrate helps to built up improved cell culture systems, optimized biomaterials and tailored surfaces for implants without the use of cost‐intensive biological components.

PP 118 MEA‐Based biosensors to study cellular differentiation and integration

The microelectrode array (MEA) technique provides functional insight into cellular communication by taking advantage of the fact that the complex interplay between different ion channels can be examined in an intact and controllable system. Due to their design, MEAs can be used for short term recording of cellular activity as well as for long term culturing, thus offering a well defined in‐vitro test system for monitoring and describing both acute characteristics and long term effects, e.g. developmental states/changes, tissue integration of foreign cells or chronic drug effects.

In our lab we have developed a biosensor based on chicken cardiomyocytes to routinely investigate cardiotoxic effects of substances known to produce arrhythmia or long QT syndrome in humans. Amplitude, shape and duration of the field action potential as well as rhythmic activity are important parameters for the characterization of the effects. Other biosensors based on pig cardiomyocyes, human heart slices and stem cell derived cardiomyocytes (hes‐CM) are under development. For some of the models the investigation is accompanied by patch‐clamp analysis to separate the underlying ional mechanisms. Additional valuable information of MEA based biosensors can be obtained by analyzing the signal transduction within the 8 × 8 electrode array providing knowledge about disturbances of the excitation spread or to serve as indicator for e.g. integration of stem cell derived cells into target tissue. Possible applications include integration of hes‐CMs into heart or stem cell derived neurons into brain tissue.

PP 119 Development of a threedimensional Trachea Cell Model

Introduction: A three dimensional model of the trachea becomes also important as an alternative for animal experiments because it shows similarities in structure and function of the human trachea.

Aim: Our aim is to develop a functional trachea test system in a bioreactor system in which the cells are cultivated under physiological conditions and simulation of the respiration. This model could be used as a test system for the analysis of different substances as an alternative for preclinical animal testing.

Materials and Methods: For the assembly we use human respiratory epithelium cells. The isolation occurs with the sprouting method from small tissue pieces of a biopsy. The cells were characterized via histological and immuno‐histological methods.

Results: In preliminary tests human respiratory cells were cultivated on inserts with a PET‐ and a biological membrane. Most of these cells are vital and show a high rate of proliferation.The cells were also cultivated in a special trachea‐reactor with simulation of the respiration to stimulate the ciliary activity to adjust the mucociliary clearance which is one of the major tasks of the trachea.

Perspectives: The cultivation of functional epithelium cells with active cinocilia and mucus secretion is very important for the development of a functional test system. Therefore a better understanding of cells and matrix and also the stimuli for the differentiation of these cells is immanently necessary.

Acknowledgments: We would like to thank the Bundesministerium für Bildung und Forschung and the WING (Werkstoffinnovationen für Industrie und Gesellschaft) for funding this project.

PP 120 Modification of biomaterial surface structures for the in vitro generation of osteochondral implants

As the fixation of engineered cartilage to bone frequently poses problems after implantation, cartilage can be generated in vitro on top of a ceramic carrier which acts as bone substitute (osteochondral implant). Until now the adhesive strength between carrier and cartilage is not sufficient to withstand high shear stresses in the human knee. Thus, the aim of this study was to investigate the influence of carrier surface structure on adhesion of chondrocytes and tissue engineered cartilage. The surface structure of hydroxylapatite carriers (Sponceram^®, Zellwerk, Germany) which was rough and inhomogeneous with local defects in the untreated state was modified by grinding with plain paper in the direction of smooth or by grinding with rough polymer sheets in the direction of structured. After characterization of the surface topography with a Charged‐coupled device camera, porcine chondrocytes were expanded on top of the carriers. After two weeks, cell density was analyzed by SEM and DAPI staining. In addition, osteochondral implants were generated according to a concept by Nagel‐Heyer et al.¹. The adhesive strength and the quality of the formed matrix were determined.

Proliferation of chondrocytes was increased fivefold on the rough surface of the untreated carrier and adhesion between carrier and engineered cartilage tissue was improved compared to the modified surfaces. Against this, a slight increase in the quality of the matrix (tissue organization and biomechanical parameters) could be observed when generated on top of the modified surfaces. In conclusion, this work delivered first promising results, which motivate for further studies.

PP 121 Porous composite materials chitosan – bioactive calcium compound particulate for bone tissue engineering

There are some disadvantages in the use of ceramics based on calcium compounds such as hydroxyapatite (HA), tricalcium phosphate (TCP), carbonated HA (CHA), and calcium carbonate (CC), as bone substitute materials. The main drawbacks are brittleness and difficulty to match ceramics exactly to the bone defect size and geometry. Composite materials based on chitosan (Ch) matrix reinforced by a ceramic particulate, being elastic, allows the overcoming above drawbacks. In this study, a method has been developed to fabricate porous composites Ch – various calcium materials such as HA, TCP, CHA, and CC used in both the powder and granulated state. The medium molecular weight Ch soluble in acetic acid at pH < 6,5 was used. The calcium materials were admixed to a water solution of Ch followed by the addition of a foaming agent that also increased the pH value of the solution. Consequently, the viscosity of the suspension was increased. The foamed mixture was frozen at −180C. After that it was soaked in ethanol to remove water and acetic acid. Porous structure of scaffolds was resulted from both the foaming by carbon dioxide and the removing of crystallized water. The scaffold's properties are dependent on the porosity of the foam, the quantity and the kind of powder and granulated fillers. The solubility of the foams was studied. Also, the biological tests in vitro were performed showing cells adhesion and viability, and absence of cytotoxicity.

PP 122 Human Oct3/4 promoter dependent lentiviral transgene expression for simplified monitoring of primate embryonic stem cell culture

Clinical application of pluripotent stem cell (SC) derivatives require cell numbers that cannot be obtained by conventional SC culture techniques. However, upscale of embryonic stem cell (ESC) culture is sophisticated and control of the differentiation status in large scale cultures by means of immunostaining and qRT‐PCR is complex and time‐consuming.

To solve this problem, we have established single cell clones of rhesus and cynomolgus monkey ESCs expressing eGFP under the control of a pluripotency‐associated promoter. Since establishment of stable transgenic clones is extremely inefficient in primate ESCs when using conventional nonviral transfection techniques, we have applied lentiviral vector technology. To this end, primate ESCs were transduced with a simian immunodeficiency virus based lentiviral construct containing the reporter gene eGFP under control of the human Oct4 promotor. Colonies of transduced cells were dissociated and single cell clones were seeded onto 96 well plates after FACS sorting or limiting dilution, respectively.

Immunocytology and qPCR demonstrated robust GFP expression in undifferentiated colonies of resulting cell clones. Moreover, gradual decrease of GFP expression during differentiation was observed by qPCR. This was confirmed by anti‐OCT3/4 staining, which also revealed good correlation to endogenous Oct3/4 expression on the single cell level. More detailed analyses concerning the eGFP expression pattern of the different cell clones during differentiation are ongoing.

The established transgenic cell clones represent a valuable tool for establishment of culture conditions for large scale production of primate ESC and induced pluripotent stem (iPS) cells as well as for reprogramming of somatic (non)human primate cells.

PP 123 Measurement of physiology and growth of cells in 3D multilayered osteochondral constructs under mechanical stimulation

With recent advances in 3D culture, metabolic gradients play an increasingly important role in cell culture and tissue engineering, effecting control over cell growth and differentiation via physiological gradients. Here, measurements of cell physiology and development were made in osteochondral constructs fabricated by a new method, where polysaccharide sponges are glued to hydroxyapatite bone scaffolds using a sol‐gel technique. The “glue” consists of silica‐sol embedded hydroxyapatite, thus mimicking the calcified layer in deep‐zone cartilage, and enables a solid, durable but bioresorbable connection between arbitrary polymeric or ceramic materials. The constructs were cultured under perfusion with or without mechanical stimulation in bioreactors from the novel labware system of the Biocer Gmbh, which allows for high replicate numbers. Scaffold permeability was measured with electrochemical sensors.

In spite of high porosity, diffusion under static conditions in a 4 mm scaffold is very low. Perfusion‐culture improved cell development, proliferation and tissue formation. Mechanical stimulation primarily enhanced cell nutrition in central regions by augmenting fluid movement, leading to an even distribution of tissue formation throughout the scaffold, which had an overall size of 15 × 8 mm. Cells grew throughout the sol‐gel layer, which formed a porous yet firm connection. Chondrocytic differentiation was not optimally induced by the chitosan scaffold, despite the application of TGF‐β3 and IGF. Based on the good results for nutrition, more differentiation‐active matrices with better mechanotransduction will be developed. The BioCer Labware Bioreactor‐System proved to be ideally suited for perfusion and mechanical stimulation of 3D tissue cultures in high replicates.

PP 124 Interaction of human bone derived cells with phospholipid coated porous titanium alloy surfaces of different morphologies

The successful use of titanium‐based implants for orthopaedic applications is sometimes hindered because of an imperfect interaction between the bone tissue and the metal surface. Porous titanium materials with suitable pore structure are good candidates for bone interaction, because they in principle permit cell “ingrowth” preventing loosening of the implant. However, the cell mobility into the pores is in many cases restricted, the cells prefer to grow on the surface. Recently it was observed for chondrocytes that they move into pores when the surface is coated by phospholipids [Willumeit et al., 2007; European Cells and Materials, 13:11‐25]. The objective of this study was therefore to create different phospholipid coated porous titanium alloy surfaces and to investigate the interactions of human bone derived cells (HBDC) with these biomimetic implant materials. Ti‐6Al‐7Nb sintered carriers with six different particle size fractions (from 45 to 500 μm) were coated adsorptive with two phospholipids (POPE and POPC). HBDC were used for the examination of cell adhesion, viability and differentiation after preincubation of phospholipid coated samples in cell medium for two hours. The structural details of the materials and interactions with growing cells were obtained by scanning electron microscopy. The data show that the viability of HBDC strongly correlates with the structure of the sintered materials. For the native materials the best results were achieved with grain sizes under 130 μm. Phospholipid coatings have a positive effect on cell‐implant interactions and improve the vitality of HBDC on samples with grain sizes above 130 μm.

PP 125 In‐Vivo tissue engineering of vascularised adipose tissue for breast reconstruction: long‐Term results and transplantation

Aim: A high demand exists for soft tissue reconstruction with adipose tissue. Especially tissue engineering of complex three‐dimensional tissue, in particular for breast reconstruction, requires vascularisation for its survival, with a view to transplanting it into the in vivo situation.The aim was to establish a technique for the de‐novo generation of large amounts of vascularised, stable and transplantable adipose tissue using a growth chamber in an animal model.

Methods: We examined the use of an vascularised adipose tissue flap (4% of the chamber volume) and a matrix scaffold in a growth chamber in the rat (1.7ml). Subsequently, we used the method in the pig with a growth chamber of 78ml volume and following pedicled transplantation of the new generated adipose tissue to simulate in‐situ breast reconstruction. Magnetic Resonance Angiography (MRA) assessed tissue growth and vascularisation within the chamber.

Results: At 12 weeks post‐insertion, the entire chamber was filled with new tissue and the adipose tissue was grown up to 60% of the chamber volume. The MRA was capable of monitoring vessel patency. Histology could confirm true hyperplasia and adipose precursor cell stimulation. No pathological changes have been seen. After pedicled transplantation, the newly generated tissue stayed stable in volume after 22 weeks.

Conclusion: The study demonstrated a promising method of producing significant amounts of vascularised, stable and transferable adipose tissue by stem cell stimulation that can be monitored non‐invasively by MRA. This is an important step towards development of a permanent autologous soft tissue replacement and a clinical application.

PP 126 Online monitoring of oxygen and pH during cell cultivation in multiwell plates

Cell cultivation requires defined in vitro conditions. This is especially important for stem cell cultivation because even small changes can have large effects on differentiation. Monitoring essential parameters like oxygen and pH enables optimization of culture conditions for complex differentiation processes.

Monitoring in small volumes was hitherto only possible through cumbersome microscoping and destructive one‐point staining tests. With the SDR SensorDish® Reader it is possible to monitor the two key parameters dissolved oxygen (DO) and pH non‐invasively and online in 24‐well multidishes. Optical sensors for DO or pH are located in each well and read out through the bottom by a small 24‐channel reader. This reader is placed in the incubator for continuous monitoring during the entire cultivation. Two applications of this system are demonstrated: DO of human embryonic stem cells under hypoxia was monitored during the entire cultivation. It was found that even short openings of the incubator door or media change had so far unconsidered effects on the DO within the sample. Furthermore, the SensorDish® Reader was used for online pH and DO monitoring in 3D cultures of chondrocytes, where examination by microscope is not possible. New insights regarding the cell activity at high concentrations were found.

PP 127 Manipulation of biological tissue and materials with ultra‐Short laser pulses at micro‐ and nanoscale

Introduction: Ultra‐short laser pulses focussed by a high numerical aperture objective create high intensities within the focal volume, which can be used to visualise as well as to manipulate materials and cells at micrometer and sub micrometer scale. The high precision achieved allows for accurate manipulation of material surfaces, tissue and single cells. The short pulse duration deposits very small amounts of energy in tissue and cells, causing only a minimum of collateral damage.

Laser microtome: The laser microtome has originally been developed for non‐invasive sectioning of biological tissue in its native state, but also allows for fast micromachining of materials used in tissue engineering. Examples are: a) structuring of Teflon discs (15 mm Ø) by cutting 1 mm × 1mm grids with line depths between 1 μm to 10 μm and a line width of 10μm b) preparation of corneal tissue for transplantation by cutting along Descemet's membrane just below the endothelium. There is also the possibility of three dimensional imaging of laser processed samples by optical coherence tomography.

Laser nanodissection system: Based on similar technique and with the fs‐laser coupled into a commercial optical microscope, this system operates in nanometer scale dimensions:

Applications of ultra‐short laser pulses offer an innovative approach to gain new insights in the field of tissue engineering and stem cell research.

PP 128 Novel Nanostructured Coatings Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells

One of the design goals of the ideal bone implant‐coating is osteoinductivity, the ability to induce the osteogenic differentiation of mesenchymal stem cells (MSC). In this study, the in vitro osteoinductive potential of novel High‐Velocity Oxygen Fuel (HVOF) ‐sprayed nano‐TiO2 + 10wt%HA (HA‐TiO2) coatings as possible mechanically superior alternatives to FDA approved atmospheric plasma spray (APS) HA coatings was evaluated. Human bone marrow mesenchymal stem cells (hMSCs) were cultured in basal and osteogenic medium (hMSC‐ob) on the novel nanostructured HA‐TiO2 and reference HA, TiO2 coatings. The novel coating was evaluated for promotion and maintenance of osteogenesis both in the presence and absence of biological stimuli through: (i) proliferation; (ii) cytoskeleton organization and cell‐substrate adhesion (F‐actin/Vinculin/ Propidium Iodide); (iii) coatings‐cellular interaction morphology and growth (SEM); (iv) biochemical markers of cellular mineralization: alkaline phosphatase (ALP) activity and osteocalcin (OC) expression; and (v) mineralization (Alizarin Red) and calcein staining, from 1 to 21 days. As seen by the increased cellular metabolism and denser fluorescence intensity on cells plated on the novel surfaces (figure 1), they induced cellular growth independently of culture conditions (osteogenic vs. unsupplemented media). The nanostructured HA‐TiO2 facilitated differentiation of cells to mature mineral producing osteoblasts as seen by the increased temporal ALP activity and OC expressions with hMSC and hMSC‐ob cultures and higher calcium deposition trend (Alizarin Red and calcein fluorescence) for the nanostructured HA‐TiO2 substrate. In conclusion, these findings collectively demonstrate the osteoinductivity of the nanostructured HA‐TiO2 coatings and their potential as novel mechanically and biologically improved load bearing implant coatings.

PP 129 Peculiarities of interaction of mouse peripheral blood cells with different passages of mouse embryonic fibroblasts

In modern cellular therapy and regenerative medicine the problem is the increase of the amount of target cells by their multiplication for introduction into an organism. As the multiplication is performed out of an organism, there is the question – whether cells remain “own” after this ex vivo procedure. The interaction of the cells from mouse peripheral blood with mouse fibroblasts prepared from 13,5 days embryo was studied. Fibroblasts at 0, 2nd and 4th passages have been tested. It has been shown that when using zero passage, as compared to control, neither morphological changes of embryonic fibroblasts after addition of white blood cells, nor interaction between them took place. Loading of peripheral white blood cells onto the monolayer of 2th passage fibroblasts resulted in appearance of changes in fibroblasts' morphology along with the presence of typical cells. Changes were expressed in cells rounding, their bulge, appearance of unevenness. Further increase of fibroblasts passages number to 4 resulted in changes, the consequence of which was vivid reaction of the added white blood cells, namely rapid and radical destroy of fibroblasts by them. These reactions cannot be connected with the remains of donor plasma at the white blood cells, because leukocytes were washed before loading. The differences revealed in the interaction of fibroblasts at different passages with the cells of peripheral blood are the evidence for the necessity of studying the interaction of cells after their multiplication out of organism with the cells of the organism.

PP 130 Test of nano‐Structured titanium dioxide coatings for use as biocompatible surfaces

A series of nanostructured TiO2 coatings on glass substrates was prepared by screen printing of TiO2 pastes and subsequent sintering at 450 °C. The surface structure was controlled on the nanoscale by the diameter of the TiO2 nanoparticles in the paste, which was chosen between 15 nm and 300 nm. For comparison, compact TiO2 layers were produced using sol‐gel and dip coating techniques. The coatings were sterilized either by autoclaving or rinsing in ethanol before cells were seeded. Glass cover slips with cells from different cell lines (epithelial, fibroblast and osteogenic origin) were incubated for 48 to 72 hours and prepared for immunocytochemistry and MTT‐assay to compare adhesion, outgrowth and proliferation. Cells`re stained for cytoskelettal proteins like tubulin and actin in combination with live stain for mitochondria and viewed with epifluorescence as most of the coatings were opaque. Differences in adhesion and growing properties of various cell lines were observed. Whereas cells on compact, dip‐coated TiO2‐layers led to similar results like controls on uncoated glass, some of the nano‐structured coatings induced improved adhesion and outgrowth. Proliferation was estimated by mitochondrial activity. Evaluation of proliferation was difficult due to hindered visibility of cell density used for the test system. Quantifying DNA by BrdU assay will avoid this restriction.

In summary, the tested cell lines showed individual effects on different nanostructured TiO2 coatings. The results demonstrate the importance of a detailed knowledge of the nanostructure and its interaction with individual cell lines to pre‐estimate effects of implants on various tissues in vitro.

PP 131 Influence of oxygen pressure on Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Fibrin Glue

Tissue engineering using biomaterials is a promising solution for cartilage replacement. The purpose of this study was to investigate whether the fibrin sealant Tissucol® provides a suitable scaffold for re‐implanting during chondrogenic replacement therapy. Pluripotent stem cells were isolated from adult human bone marrow (hMSCs), cultured and characterised by FACS (CD105+/CD106+, CD45‐/CD14‐/CD34‐), by differentiation‐assays as well as by Oct4‐expression profile. Using appropriate preparation and applying centrifugal forces leads to a reproducible large‐holed porous 3D network in which the hMSCs are homogenously distributed and that allowed hMSCs to survive throughout the period of culture in either proliferation or chondrogenic differentiation medium under normoxic (21% O2) or hypoxic (3% O2) conditions. Morphology (as determined by light‐ and electron microscopy) and proliferation (Ki67 staining) of the embedded hMSCs did not markedly vary under normoxic and hypoxic culture even after 21 days in culture. The stem cell marker Oct4 was expressed during the whole culture period. Under chondrogenic differentiation conditions, especially under hypoxic conditions, we observed rounded chondrocyte‐like cell types and a chondrogenic phenotype assessed by mRNA expression of collagen II and Alcian blue staining. hMSCs seeded into large‐holed porous preparations of Tissucol® survive, proliferate and keep their stem cell character. Furthermore, culturing the cells in corresponding medium induces chondrogenic differentiation, which could be remarkably and significantly enhanced under hypoxic conditions. This firstly qualifies Tissucol® as adequate scaffold for cartilage generation and secondly suggests benefits in emulating the bradytrophe environment of cartilage in vivo by studying chondrogenesis in vitro under hypoxic culture conditions.

PP 132 A novel collagen cell carrier for cell culture systems

The biological function of complex three‐dimensional cell culture systems strongly depends on the chemical and mechanical properties of the cell carriers employed. Numerous biocompatible materials have been modified to optimize cell attachment, integration and cellular differentiation processes in vitro and in vivo. In this study, we introduce a novel bovine collagen I‐based cell carrier which was tested in different cell culture systems. This scaffold exhibits high mechanical stability coupled with material thicknesses below 20 μm, placing it in an interesting range for the generation of complex multilayered tissue constructs. Various cell culture populations were investigated to evaluate the biocompatibility of the collagen scaffold in vitro and its impact on cell viability, proliferation and differentiation. Cell culture experiments with the human osteosarcoma cell line Saos‐2 did not show significant differences in viability and cell proliferation between cells seeded on conventional uncoated culture plates and those grown on the collagen scaffold. Differentiating murine osteoblasts and human mesenchymal stem cells strongly calcified the collagen scaffolds under osteoinductive cell culture conditions. Primary cardiomyocytes from fetal murine hearts seeded on the collagen cell carrier maintained their spontaneous contractile activity in culture over the entire 7‐day observation period. Both the mechanical characteristics and the demonstrated in vitro biocompatibility of this collagen I carrier highlight its suitability for in vitro studies and in vivo applications in basic research and in the field of Regenerative Medicine.

PP 133 Cell seeding densities affect differentiation of human adipose tissue‐Derived stem cells – Do connexins play a role?

Adipose tissue‐derived stem cells (ASC) are a promising cell type for regenerative medicine. ASC are relatively easy to isolate and propagate and could thus be utilized for stem cell therapies. However, knowledge about ASC characteristics is fragmentary. In this study, we examined the effects of seeding densities on the differentiation of ASC. ASC were seeded in different densities to control the extent of intercellular contacts. The cells were then exposed to adipogenic or osteogenic differentiation stimuli. Proliferation as well as adipogenic or osteogenic differentiation characteristics were followed for two weeks. To examine the impact of intercellular contacts, in particular connexins, on ASC differentiation, the experiments were also performed in presence of inhibitors for connexin signalling. After two weeks of cultivation, the initial differences in cell density were nearly balanced. Whereas osteogenic stimulation generally led to increased proliferation (doubled cell numbers compared to non‐stimulated cells), adipogenic stimulation led to reduced proliferative activity (ca. 30% less cells). Adipogenic differentiation was strongly dependent on the initial seeding density, in that only those cultures that were confluent from the beginning (highest seeding density) showed significant adipogenic differentiation. This is in contrast to osteogenic differentiation which was slightly increased by sparser cell densities. Connexin inhibitors did not show a significant influence on differentiation degree. Thus, primary seeding densities influenced the degree of adipogenic and osteogenic differentiation and preliminary results do not indicate an involvement of connexin signalling herein. This work was financed by EU and the Ministry of Economic Affairs, Employment and Tourism Mecklenburg‐Vorpommern.