Jürgen H. Fischer

Degradation of poly(D, L)lactide implants with or without addition of calciumphosphates in vivo

The study was aimed at examining the in vivo degradation of pure poly(D,L)lactide (PDLLA) or PDLLA with an admixture of calciumphosphates. One rod (20 x 3 x 2 mm) and one cube (3 x 2 x 2 mm) of pure PDLLA, PDLLA with tricalciumphosphate (PDLLA + TCP) or PDLLA with calciumhydrogenphosphate (PDLLA + CHP), respectively, were implanted into the dorsal muscles of 50 male Wistar Albino rats. After definite intervals (from 2nd to 72nd week), pH measurements were performed in the environment of the implants. Afterwards, the cubes with their surrounding tissues were excised for histological examinations, measurements of the outer dimensions and mechanical analyses of the explanted rods were performed. No drop of more than 0.1 pH units was detectable in the tissue surrounding any type of implants. No advantageous effect of the calciumphosphates could be proved. A mild foreign body reaction could be observed around PDLLA implants. After 72 weeks, pure PDLLA had been totally resorbed from the extracellular space, the degradation of calciumphosphate-enriched PDLLA was still in progress. A large amount of inflammations occurred in the tissues surrounding PDLLA with an admixture of slowly degrading TCP or CHP, leading to two abscesses and four fistulas at PDLLA + TCP, and two abscesses and three fistulas at PDLLA + CHP implantation site. Bending strength of pure PDLLA was constant up to the 4th week post-implantation and reduced to 60% of the initial value up to the 12th week. No traces of crystallinity could be observed during the degradation of PDLLA. As a conclusion of the study, complete resorption from the extracellular space and tissue tolerance of pure PDLLA is proved. An admixture of small calciumphosphate particles is not suitable to improve the biocompatibility of PDLLA but leads to a decrease in the mechanical characteristics.

Poor Cell Survival Limits the Beneficial Impact of Mesenchymal Stem Cell Transplantation on Acute Kidney Injury

Background: Although renal tubular epithelium has a great capacity for repair it has been suggested that the administration of mesenchymal stem cells may accelerate the recovery following severe ischemic injury. Methods: Here we analyzed the survival rate and organ distribution of transplanted mesenchymal stem cells as well as their contribution to kidney regeneration after ischemic renal injury using functional tests, histological examination as well as quantitative real-time PCR. Results: Intravenously injected stem cells were mainly trapped in lungs and liver. One hour after injection, less than 1% of the injected stem cells could be detected in the injured kidneys. These cells disappeared within the first few days and did not replace renal epithelial cells precluding substantial transdifferentiation. To clarify whether reinforced stem cell delivery might promote sustained survival or conversion to tubular epithelia, stem cells were directly injected into the injured kidneys. Although these grafted cells also did not show sustained survival or contribute to structural renal repair, stem cell injection was associated with a significant but transient initial decrease in serum creatinine. Conclusion: These data suggest that mesenchymal stem cells do not significantly contribute to epithelial renewal after ischemic injury, promoting the idea that the major impact of cell-based therapy for acute kidney injury may result from paracrine or endocrine effects unrelated to stem cell transdifferentiation.

Role of balloon occlusion for mononuclear bone marrow cell deposition after intracoronary injection in pigs with reperfused myocardial infarction

AIMS 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 (IM) injection. METHODS AND RESULTS Four hours after LAD ligation in male pigs, reperfusion was allowed (confirmed by coronary angiography). Five days later, 1 x 10(8) autologous (111)Indium-labelled MNC were injected IC/noB (n = 4), IC/B (n = 4), or IM (n = 4). At 1 h the fraction of injected MNC that was detected in the heart was 4.1 +/- 1.1% after IC/noB injection, 6.1 +/- 2.5% after IC/B injection (P = 0.19), and 20.7 +/- 2.3% after IM injection (P < 0.001 vs. IC/noB and IC/B). At 24 h it was 3.0 +/- 0.6% (IC/noB), 3.3 +/- 0.5% (IC/B, P = 0.43), and 15.0 +/- 3.1% (IM, P < 0.001 vs. IC/noB and IC/B). Dynamic scintigrammes during each of four consecutive IC/B injections showed a rapid 19.6 +/- 8.0% cell loss during balloon inflation (no-flow period, phase 1) and a rapid 36.6 +/- 17.8% cell loss after balloon deflation (re-flow period, phase 2). After each of four consecutive IC/noB injections the peak cell deposit was lower, followed by one phase of rapid cell loss (30.9 +/- 11.0% after 6 min). After IM injection only a slow linear cell loss was observed (9.7% per h). In histology, PKH-67 labelled cells only rarely had passed the endothelial barrier after 24 h after IC injection, while they were exclusively found in the interstitium after IM injection. CONCLUSION The observation of a similar cell persistence after IC 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 persistence of cells plays a role for the clinical benefit of cardiac cell therapy, IM injection may be superior to IC applications.

A simple method for orthotopic liver transplantation in the rat. Cuff technique for three vascular anastomoses.

A simple method for orthotopic liver transplantation in the rat is described in detail. A cuff technique was applied to all anastomoses of the supra- and infrahepatic venae cavae and the portal vein. It simplified and shortened the implantation of the graft as well as eliminating the need for a microvascular suture technique. In the last series of 20 transplants, the survival rate was 85% after 1 week and 55% after 2 months with normal hepatocellular function.

Respiratory chain deficiency slows down cell‐cycle progression via reduced ROS generation and is associated with a reduction of p21CIP1/WAF1

We have used HeLa cells without mitochondrial DNA (ρ0‐cells) and transient ρ0‐phenocopies, obtained from wild‐type cells by short‐term treatment with ethidium bromide, to analyze how the absence of a functional mitochondrial respiratory chain slows down proliferation. We ruled out an energetic problem (ATP/ADP content) as well as defective synthesis of pyrimidine, iron‐sulfur clusters or heme as important causes for the proliferative defect. Flow cytometric analysis revealed that reactive oxygen species were reduced in ρ0‐cells and in ρ0‐phenocopies, and that, quite unusually, all stages of the cell cycle were slowed down. Specific quenching of mitochondrial ROS with the ubiquinone analog MitoQ also resulted in slower growth. Some important cell‐cycle regulators were reduced in ρ0‐cells: cyclin D3, cdk6, p18INK4C, p27KIP1, and p21CIP1/WAF1. In the ρ0‐phenocopies, the expression pattern did not fully duplicate the complex response observed in ρ0‐cells, and mainly p21CIP1/WAF1 was downregulated. Activities of the growth regulatory PKB/Akt and MAPK/ERK‐signaling pathways did not correlate with proliferation rates of ρ0‐cells and ρ0‐phenocopies. Our study demonstrates that loss of a functional mitochondrial electron transport chain inhibits cell‐cycle progression, and we postulate that this occurs through the decreased concentration of reactive oxygen species, leading to downregulation of p21CIP1/WAF1. J. Cell. Physiol. 209: 103–112, 2006.

pH-stabilization of predegraded PDLLA by an admixture of water-soluble sodiumhydrogenphosphate--results of an in vitro- and in vivo-study.

Aim of the study was to examine if the addition of buffering sodiumhydrogenphosphate to poly(D,L)lactide(PDLLA) would stabilize the pH-value in the in vivo environment of implanted material and whether this improves its biocompatibility. The material was predegraded just to the point of viscous disintegration to test the PDLLA in the moment of its most aggressive effect on the surrounding tissue. Racemic amorphous PDLLA was injection-molded with or without the admixture of 1 mol NaP per 100 mol lactate, the degradation product of PDLLA (=1 mol%) to form 20mm x 3 mm x 2mm rods. Predegradation was performed by storing the rods at 55 degrees C for 14 days, just to the point of beginning dissolution. Predegraded PDLLA or PDLLA + NaP samples were used for in vitro incubation tests, as well as for the in vivo study, where the rods were implanted into the spinal muscles of 30 male Wistar rats. Repeatedly, measurements of the pH-value were made in the incubation solutions in vitro. The surrounding tissue of the implanted samples as well as the normal contralateral muscle tissue was checked for its pH-value in a group of 3 rats, respectively, anaesthesized at various time intervals after implantation. After these measurements the implants and their surrounding tissues were excised for histological examination. In Ringers solution pH-values dropped immediately within the first week of incubation of both predegraded materials reaching -4 pH units after 4 weeks in the PDLLA containing medium, after 6 weeks in the PDLLA + NaP containing medium. Soerensen buffer slowed the pH decrease with significant differences between the material groups up to the 28th week. In vivo, the pH of the surrounding tissue was influenced by the implanted PDLLA material up to the 4th week, while the admixture of NaP resulted in a significant pH stabilization. A higher quantity of macrophages and giant cells were seen between the 2nd and 6th week after the implantation in the environment of pure PDLLA compared with PDLLA + NaP. Complete resorption of predegraded pure PDLLA or PDLLA + NaP from the extracellular space was reached 28 weeks postimplantation in vivo. Thus, sodiumhydrogenphosphate improves the biocompatibility of degrading PDLLA at the point of viscous disintegration by stabilizing the pH-value in the environment of the implants for several weeks and reducing adverse tissue reactions.

Transplantation of De Novo Scaffold-Free Cartilage Implants Into Sheep Knee Chondral Defects

Background New cell-based treatments for articular cartilage repair are needed. As the optimal scaffold for cartilage repair has yet to be developed, scaffold-free cartilage implants may remove the complications caused by suboptimal scaffolds. Hypothesis The implantation of a scaffold-free, autologous de novo cartilage implant into standardized full-thickness cartilage defects of femoral condyles in sheep leads to a qualitatively better regenerative tissue than does periosteal flap alone or no treatment. Study Design Controlled laboratory study. Methods Chondral defects 4 mm in diameter (1 per sheep) were created in the center of 1 medial femoral condyle of 48 sheep. Twelve defects were allowed to heal spontaneously, 16 defects were covered with periosteal flaps alone, and 20 defects were filled with autologous de novo cartilage graft and overlaid with a periosteal flap. Differences were assessed macroscopically using the International Cartilage Repair Society score and microscopically using the International Cartilage Repair Society histological score and Mankin score at 26 and 52 weeks. Results The results of the International Cartilage Repair Society Cartilage repair assessment showed that the transplant group was better than was the untreated control at both time periods but not significantly different than was the periosteal flap group. Implanted groups demonstrated a marked improvement in grade of defect filling, cartilage stability, cell distribution, and matrix assessments in each method of assessment. In the transplant group, 2 defects were filled with hyaline cartilage, 5 with mixed hyaline and fibrocartilage, and 2 with fibrocartilage alone. Conclusion Chondral defects treated with de novo cartilage transplantation show qualitatively better microscopic and macroscopic regeneration than do those treated with periosteal flaps alone. Clinical Relevance Results of the current study show that third-generation autologous chondrocyte transplantation is a promising development in the field of biologic cartilage regeneration. Future studies should compare this technique with the original Brittberg technique.

Effectivity of freshly prepared or refreshed solutions for heart preservation versus commercial EuroCollins, Bretschneider's HTK or University of Wisconsin solution.

Rat hearts were preserved for 18 hr under totally ischemic storage conditions at 0–1°C with the commercially supplied EuroCollins, Bretschneiders HTK, and University of Wisconsin (UW) preservation solutions compared with our new preservation solution, EuroFlush-glutathione solution, and a “refreshed” UW solution (UWG) with 3 mmol/L reduced glutathione added before use. Recovery of the organs was measured during 30 min of parabiotic reperfusion with whole blood of a host rat of the same inbred LEW strain, following an initial warm reflush for 5 min. Functional measurements were performed using a latex balloon in the left ventricle. The metabolic recovery was determined from the myocardium freezeclamped at the end of reperfusion. The left ventricular pressure (LVP) amplitude during pacing to a heart rate of 300/min, as well as +dp/dtmax, —dp/dtmax, isotonic stroke volume, coronary flow, ATP, and ECP values, recovered significantly better after storage in Euro-Flush-glutathione solution (LVP: 63% of controls on average) compared with when the commercially available solutions were used (EuroCollins: 20%, HTK: 42% of controls in LVP). Hearts preserved in UW solution ViaSpan did not recover during the reperfusion period, when unfiltered solution was used. Filtered ViaSpan resulted in LVP recoveries of 38% of controls, while addition of reduced glutathione immediately before use (UWG) improved the effectivity of this solution significantly (LVP 63% of controls). Similar improvements were found for all other functional and metabolic parameters. Thus, the effectivity of UW solution ViaSpan depends upon extraction of the typical particles by a filtering procedure. Effectivity can be improved by a refreshment procedure with reduced glutathione immediately before use.

Experimental bile duct protection by intraductal cooling during radiofrequency ablation.

The use of radiofrequency ablation (RFA) for liver tumours is limited by the proximity of large bile ducts to the targeted lesion. The aim of this randomized study was to evaluate intraductal cooling as a mean of protecting the bile ducts during RFA.

In-vivo-Untersuchung zur Degradation von Poly-(D,L-)Laktid- und Poly-(L-Laktid-co-Glykolid)-Osteosynthesematerial

Ziel der Untersuchung war die vergleichende histologische Untersuchung der Degradation von Poly-(D,L-)Laktid (Resorb X®) und Poly-(L-Laktid-co-Glykolid) (LactoSorb®) in vivo. Bei 26 Chinchilla-Kaninchen wurden je eine LactoSorb®- und Resorb-X®-Osteosyntheseplatte mit entsprechenden Schrauben an den lateralen Femurflächen beidseits fixiert. Nach der Injektion von Fluorochromen zur intravitalen Sequenzmarkierung wurden die Femora nach 1, 6, 12, 14, 16, 21 und 26 Monaten entnommen und die Schrauben-Platten-Knochen-Präparate röntgenologisch, fluoreszenz- sowie lichtmikroskopisch untersucht. Im ersten Monat nach der Implantation war eine Knochenneubildung kranial und kaudal der Polymere sowie entlang der Schraubenwindungen erkennbar. Nach 6 Monaten waren die Implantate vollständig von Knochen bedeckt. Während sich die LactoSorb®-Schrauben polarisationsoptisch unverändert als doppelbrechende Struktur zeigten, trat beim Resorb X® eine von außen nach innen fortschreitende Resorption durch phagozytierende Knochenmarkzellen auf. 12 Monate postoperativ war das Resorb-X®- und nach 14 Monaten das LactoSorb®-Osteosynthesematerial lichtmikroskopisch vollständig resorbiert und durch Knochenmarkzellen ersetzt. Die Knochenumbauvorgänge hielten auch 26 Monate nach der Implantation noch an. Die Resorption von Resorb X® war eher beendet als die von LactoSorb®. Bei beiden Materialien konnte lichtmikroskopisch eine vollständige Resorption nach 12 bzw. 14 Monaten festgestellt werden, die Ossifikation der Bohrlöcher war dagegen auch nach 26 Monaten noch nicht abgeschlossen. Comparison of the degradation of poly(D,L)lactide (Resorb X®) or poly(lactide-co-glycolide) (LactoSorb®) in vivo. LactoSorb® and Resorb X® osteosynthesis plates were fixed at the lateral aspect of the femora of 26 Chinchilla rabbits using the respective osteosynthesis screws. After intraperitoneal injection of fluorochromes the screw plate bone blocks were resected after 1, 6, 12, 14, 16, 21, 26 months and radiologic, histologic as well as fluorescence microscopic examinations were carried out. Newly formed bone was detectable above and beneath the polymers 1 month after the implantation. The implants were totally covered by newly formed bone after 6 months. While the LactoSorb® screws were found to be as birefringent as after 1 month, in the Resorb X® screws a continuous resorption by phagocytizing marrow cells starting from the periphery was detectable. Resorb X® was totally resorbed in histologic slides 12 months after implantation, while total resorption of LactoSorb® lasted 14 months; both polymers were replaced by marrow cells. Bone remodeling was not finished 26 months after implantation in both polymers. Resorption of Resorb X® was finished earlier than the resorption of LactoSorb®. Both materials were found by fluorescence microscope to be completely resorbed after 12 or 14 months, but bone remodeling of the screw holes was not yet finished 26 months after implantation.