Lutz Wilhelm

Morphometric immunohistochemical examination of the inflammatory tissue reaction after implantation of calcium phosphate-coated titanium plates in rats

Calcium phosphate (CaP) preparations are established coatings for titanium-based medical implants used for bone reconstruction. However, biodegradation of the coating can result in microparticles that subsequently cause inflammatory reactions. The present study was therefore aimed at investigating the inflammatory response to two series of CaP-coated titanium plates: Ti-brushite (Ti-B) and Ti-hydroxyapatite (Ti-H) implants. Fifteen male LEW.1A rats received one plate of each series and a pellet (5 x 2 mm) of sol-gel derived silica/CaP (SCP implants) implanted into the back musculature. After 7, 14 and 28 days, five rats were killed and the implants were removed with the surrounding tissue. Quantitative immunohistochemistry was performed on frozen sections. Total monocytes/macrophages, tissue macrophages, T-cells, MHC-class-II-positive cells and proliferating cells were counted. For the Ti-B implants, the number of monocytes/macrophages remained constant while the other cell populations increased. In contrast, for the Ti-H implants the number of monocytes/macrophages decreased while the other cell populations remained constant. The SCP implants demonstrated degradation and scattering into smaller particles with an increase for all cell populations except the proliferating cells. Human mesenchymal stem cells demonstrated adherence and a flat morphology on Ti-B and Ti-H implants and no remarkable difference between both implants. Taken together, the in vivo data demonstrate that the short-term inflammatory response against a hydroxyapatite coating is lower in comparison to a brushite coating, and that the morphology of cells growing in vitro is similar on both layers.

Pathophysiological Measurement and Results After Laparoscopic Fundoplication for Gastroesophageal Reflux Disease

Abstract.Purpose: Both surgical and conservative treatments for gastroesophageal reflux disorder (GERD) are controversial. The aim of this prosepective study was to examine outcomes after laparoscopic antireflux surgery. Methods: The subjects were 143 patients who underwent laparoscopic antireflux surgery. Following diagnostic procedures 126 patients were allocated to a total fundoplication group (360°C, Nissen-DeMeester) and 17, to a posterior semifundoplication group (250–270°, Toupet). All complications were registered, and pathophysiological and outcome data were examined 3, 6, and 9 months after surgery. Results: By 6 months after surgery the mean lower esophageal sphincter (LES) pressure had improved significantly, to 14.8 mmHg in the Nissen-DeMeester group, and to 12.1 mmHg in the Toupet group, corresponding to successful prevention of esophageal reflux in both groups. Dysphagia was more common in the early postoperative period after total fundic wrap (17% vs 12%), but this difference disappeared in time. All patients reported complete relief of reflux symptoms, although two of those who underwent the Nissen-DeMeester fundoplication experienced relapse of GERD and required open reconstruction (1.4%). The laparoscopic procedure was converted to open surgery in three patients (2%). There were no associated deaths and the perioperative complication rate was 4.2%. Conclusion: Laparoscopic antireflux surgery is an effective treatment for GERD. More than 93% of the patients in this series rated their outcome as good to excellent following the operation.

Digital image analysis for morphometric evaluation of tissue response after implanting alloplastic vascular prostheses.

The aim of this study was to examine the suitability of digital image analysis, using the KS400 software system, for the morphometric evaluation of the tissue response after prosthesis implantation in an animal model. Twenty-four female pigs aged 10 weeks were implanted with infrarenal Dacron(R) prostheses for 14, 21, 28, and 116 days. Following the explantation and investigation of the neointima region, the expression of beta-1-integrin, the proliferation rate by means of Ki-67 positive cells, and the intima thickness were evaluated as exemplary parameters of the tissue response after implantation. Frozen tissue sections were immunohistochemically stained and subsequently examined using computer-aided image analysis. A maximum expression of 32.9% was observed for beta-1-integrin 14 days after implantation, gradually declining over time to 9.8% after 116 days. The proliferation rate was found to be 19% on day 14, increasing to 39% on day 21 with a subsequent gradual decline to 5% after 116 days. The intima thickness increased from 189.9 microm on day 14 to 1228.0 microm on day 116. In conclusion, digital image analysis was found to be an efficient and reproducible method for the morphometric evaluation of a peri-prosthetic tissue response.

Antigenität von Polyestergefäßprothesen (Dacron)

Zusammenfassung Bisher stehen keine komplett inerten Biomaterialien zur Verfügung und es existiert kein universeller Test zur Objektivierung der Biokompatibilität. Dies resultiert aus der individuellen Variabilität des Empfängerorganismus, insbesondere hinsichtlich der entzündlichen Reaktionsbereitschaft. Auch nach Implantation von Gefäßprothesen aus polymeren Biomaterialien kommt es zu einem chronischen Entzündungsprozeß. Dieser führt ursächlich durch Hydrolyse oder Autoxidation zur Biodegradation des Implantats. Mit unseren Untersuchungen galt es, eine möglicherweise bestehende, spezifische humorale Immunantwort nach Implantation von Segmenten einer kollagenimprägnierten Polyesterprothese (Dacron) in einem Tiermodell darzulegen. Balb/c-Mäusen wurde am 1., 18., 38. und 290. Versuchstag ein Prothesensegment intraperitoneal implantiert. Die Bestimmung der Serumantikörper erfolgte mit einem modifizierten Enzymimmunoassay unter Verwendung der Prothese als Target. Spezifische Antikörper gegen Polymere wurden nach wiederholter Implantation bei allen Tieren bis zum 322. Versuchstag nachgewiesen. Dabei konnte eine Antikörperbildung gegen die Kollagenimprägnierung ausgeschlossen werden. Die Antikörperbildung wurde durch den Zusatz von komplettem Freund-Adjuvans in Verbindung mit der ersten Implantation verstärkt. Der Nachweis von spezifischen Antikörpern gegen Polymere könnte zukünftig ein Parameter zur Testung der Biokompatibilität darstellen.Abstract At present there is neither an completely inert biomaterial available, nor does a universal test exist which objectively evaluates biocompatibility. One reason is the individuality of the host, especially with regard to the inflammatory response. Inflammation was found to induce biodegradation by hydrolysis or auto-oxidation of vascular prosthetic matrix after implantation. The present study was performed to investigate the specific humoral immune response after implantation of segments of a collagen-impregnated polyester prosthesis (Dacron) in Balb/c mice on experimental days 1, 18, 38, and 322. Serum antibody detection was performed by modified enzyme immunoassay using the prosthesis as a target. Specific polymer antibodies, enhanced by repeated implantations, could be detected in all mice which received grafts up to experimental day 322. These antibodies were not directed against the collagen coating. The antibody formation could be further enhanced by a combined administration of complete Freunds adjuvant together with the first implantation at experimental day 1. Results suggest that specific polymer antibodies, reflecting an inflammatory response, might be an additional parameter for biocompatibility testing of vascular prostheses.

Time course of fibronectin in the peri-implant tissue and neointima formation after functional implantation of polyester-based vascular prostheses with different porosity in pigs

Intima hyperplasia, resulting from extracellular matrix (ECM) secretion, can lead to vascular prosthesis occlusion and is a major problem in vascular surgery. Fibronectin might contribute to ongoing ECM secretion. However, the exact role of fibronectin and its influence on neointima formation remains unclear. This study was aimed at investigating the time course of the fibronectin area fraction and neointima formation following the functional implantation of three different polyester vascular prostheses into pigs. The infrarenal aorta from 15 animals (n = 5/group) was replaced by prosthesis segments with low, medium and high primary porosity. After 7, 14, 21, 28 and 116 days, the prostheses were morphometrically examined. Overall, the fibronectin area fraction was inversely correlated with the neointima thickness, demonstrating high fibronectin levels in the early phase (days 7 and 14) and low levels in the later phase with almost complete neointima formation (days 21-116). Throughout the study, fibronectin levels were highest at the proximal anastomosis region. The low porosity prosthesis had the highest fibronectin area fraction and a delayed neointima formation in the middle phase (days 21 and 28) but the highest neointima lining on day 116. The results indicate a relationship between fibronectin and neointima formation with the prosthesis porosity, demonstrating the importance of the textile design for tissue reactions following implantation.

Der Einfluss der Oberflächen-Beschichtung auf die humorale Immunreaktion gegen implantierte Polyester-Gefäβprothesen im Tiermodell

Foreign-body reaction is an obligatory subsequence of biomaterial implantation whose extent can be determined on the one hand from the implant surface, and on the other through individual factors. This study investigates individual, humoral immune response in LEW.1A rats after repeat i.m. implantation of commercially available polyester vascular prostheses coated with different substances (collagen = G1, gelatin = G2, human serum albumin = G3) as well as from a poly-tetrafluoroethylene control graft prosthesis (G4). Sham procedures in control animals were designated G5. Serum sample Ab detection was performed by enzyme immunoassay employing polymer particles of a non-impregnated polyester prosthesis as a target. In contrast to the G4 & G5 groups, all animals with polyester implants demonstrated a differentiated anti-polymer Ab development (G2 > G3 > Gl). Polymer Ab prevalence was 100% for G2 + G3 on day 22. For Gl it was a maximum of 71.4%. Only G3 revealed a specific Ab reaction to the surface-coating as well as the strongest overall immunological reaction. G4 demonstrated no specific Ab development against polytetrafluoroethylene. Our results have showed a specific immune reaction which varied not only as a result of the applied polymer, but also due to the surface-coating as well as individual factors.

Differenzierung der humoralen Immunantwort gegen Bestandteile von Kollagen-imprägnierten Dacron-Gefäßprothesen im Tiermodell Schwein

Background/Aim: Recent studies have shown the induction of specific antibodies after repeated implantation of polymeric biomaterials such as Dacron vascular prostheses in rats and mice. The present study was aimed at further differentiating the humoral immune response against components of prostheses after single functional implantation of Dacron vascular prostheses in pigs. Methods: The infrarenal aortas of 22 female pigs weighing 24-29 kg were resected and replaced by a segment of a collagen-impregnated Dacron vascular prosthesis (Meadox hemashield, length 5 cm, diameter 0.8 cm). Serum samples were drawn at days 1 (day of implantation), 10,17,24,62,116. Detection of polymer antibodies was performed by modified enzyme immunoassay using the prosthesis as an antigenic target. Furthermore, antibodies against the coating substance bovine collagen and serum IgG content of samples were determined by ELISA. Results: A significant antibody formation against the polymer matrix was already determined at experimental day 10 (p<0.05 vs day 1). The highest antibody prevalences were obtained at days 10 and 17 with 40.9% (9/22) antibody-positive animals. Circulating polymer antibodies were detected in 36.4% (8/22) of pigs at day 62, whereas only 3/22 had polymer antibodies at day 116. The prevalence of antibodies against native bovine collagen varied between 40.9% (9/22 animals) at day 24 and 68.2% (15/22 animals) at experimental day 116. No correlation was found between polymer antibodies and collagen antibodies as well as time course of serum IgG concentration. Conclusion: IgG antibodies against the prosthesis matrix and the prosthesis impregnation were induced after single functional implantation of a Dacron vascular prosthesis in a pig animal model. The time course of antibody formation as well as antibody titers have shown a high variability in individual animals. The detection of specific antibodies against components of implants reflecting the individual inflammatory response might be a parameter of individual bio(in)compatibility and in appropriate vascular prosthesis selection.