D. Rickert

Biocompatibility testing of novel multifunctional polymeric biomaterials for tissue engineering applications in head and neck surgery: an overview.

Biomaterial research and tissue engineering are rapidly growing scientific fields that need an interdisciplinary approach where clinicians should be included from the onset. Biocompatibility testing in vitro and in vivo comprise the agarose-overlay test, the MTT test, direct cell seeding tests and the chorioallantoic membrane test for angiogenic effects, among others. Molecular biology techniques such as real-time polymerase chain reaction and microarray technology facilitate the investigation of tissue integration into biomaterials on a cellular and molecular level. The physicochemical characterization of biomaterials is conducted using such methods as X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Excellent biocompatibility and biofunctionality were demonstrated for a series of recently developed multifunctional biodegradable, polymeric biomaterials both in vitro and in vivo. Novel, multifunctional polymeric biomaterials offer a highly specific adjustment to the physiological, anatomical and surgical requirements and can thereby facilitate new therapeutic options in head and neck surgery.

[Cell proliferation and cellular activity of primary cell cultures of the oral cavity after cell seeding on the surface of a degradable, thermoplastic block copolymer].

Using standard cell biological and biochemical methods we were able to test the ability of a degradable, thermoplastic block copolymer to support the adhesion, proliferation, and the cellular activity of primary cell cultures of the oral cavity in vitro. The delicate balance between a group of endogenous enzymes, Matrix Metalloproteinases (MMPs), and their inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during processes like wound healing or the integration of biomaterials in surrounding tissues after implantation. Recently developed, biodegradable thermoplastic elastomers with shape-memory properties may be the key to develop new therapeutical options in head and neck surgery. Primary cell cultures of the oral cavity of Sprague-Dawley rats were seeded on the surface of a thermoplastic block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells were analyzed for MMPs and TIMPs after different periods of cell growth. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. No statistically significant differences in the appearance and the kinetic of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the polymer surface compared to the control. An appropriate understanding of the molecular processes that regulate cellular growth and integration of a biomaterial in surrounding tissue is the requirement for an optimal adaptation of biodegradable, polymeric biomaterials to the physiological, anatomical, and surgical conditions in vivo to develop new therapeutic options in otolaryngology and head and neck surgery.Ein Schwerpunkt in der Regenerativen Medizin liegt auf der Wiederherstellung der physiologischen Funktion von Geweben oder Organen. Dies kann durch künstliche Implantate oder durch Gewebebzw. Organtransplantationen erreicht werden. Aufgrund der demographischen Entwicklung westlicher Industriegesellschaften wird der Bedarf an Gewebeund Organersatz in den nächsten Jahren weiter ansteigen. Gewebeund Organtransplantationen sind u. a. mit dem Risiko von immunologischen Reaktionen, der Notwendigkeit einer Immunsuppression beim Empfänger sowie einer limitierten Verfügbarkeit von Spendergeweben und –organen assoziiert [14]. In der Hals-Nasen-Ohrenheilkunde und Kopf-Halschirurgie können progressive Erkrankungen sowie therapeutische Interventionen, insbesondere nach tumorchirurgischen Resektionen, mit der Destruktion und/oder dem Verlust von Geweben assoziiert sein. Während an Fragestellungen zum klinischen Einsatz von Biomaterialien in operativen Fächern wie u. a. der Orthopädie, der Herzchirurgie, der Augenheilkunde oder der Urologie schon seit Jahren intensiv gearbeitet wird, steht in der Hals-Nasen-Ohrenheilkunde und der Plastischen Kopf-Halschirurgie die Entwicklung und Anwendung von biokompatiblen Werkstoffen noch am Anfang. Die Rekonstruktion von Geweben und Organen des oberen Aerodigestivtraktes mit adäquater Funktion stellt eine große wissenschaftliche und klinische Herausforderung für die Zukunft dar.

Erste Ergebnisse zur Untersuchung der Stabilität und Gewebeintegration eines abbaubaren, elastischen Copolymers im Tiermodell / First results of the investigation of the stability and tissue integration of a degradable, elastomeric copolymer in an animal model

Zusammenfassung Im Tiermodell wurden die mechanische Stabilität und die Dichtigkeit des Polymer-Gewebe-Verschlusses eines neuartigen, abbaubaren, elastischen Copolymers untersucht. Um das Biomaterial unter extremen chemischen, enzymatischen und mechanischen Bedingungen zu untersuchen, wurde das Material zur Rekonstruktion eines durchgreifenden Magenwanddefektes bei Sprague-Dawley-Ratten verwendet (n=42). In der Kontrollgruppe (n=21) wurde ein primärer Wundverschluss des identischen Magenwanddefektes ohne Biomaterialimplantation durchgeführt. In der Baselinegruppe (n=21) wurden die Versuchstiere unter identischen Versuchsbedingungen ohne operativen Eingriff gehalten. Die Versuchsdauer betrug 1 Woche, 4 Wochen und 6 Monate. Das Gewicht der Versuchstiere wurde präoperativ und vor der Explantation bestimmt. Im Rahmen der Explantation erfolgte eine Druckmessung im Magen nach maximaler Aufdehnung durch Luftinsufflation, um die Dichtigkeit des Verschlusses zwischen Polymer und Magenwand nachzuweisen. Nach einwöchiger Implantations- bzw. Versuchsdauer zeigte sich eine statistisch signifikante Gewichtszunahme nur in der Baselinegruppe. 4 Wochen und 6 Monate nach dem bauchchirurgischen Eingriff fand sich in allen drei Gruppen eine statistisch signifikante Gewichtszunahme im Vergleich zum präoperativen Gewicht der Versuchstiere. Gastrointestinale Komplikationen wie Fistelbildungen, Perforationen oder Peritonitiden traten bei keinem Versuchstier auf. Die intragastrale Druckmessung nach maximaler Aufdehnung des Magens ergab keine statistisch signifikanten Differenzen in der Implantat-, der Kontroll- und der Baselinegruppe zu den drei untersuchten Zeiträumen. Die Dichtigkeit des Nahtverschlusses zwischen dem Polymer und dem umgebenden Gewebe war bei allen Tieren der Implantatgruppe nachweisbar. Die erforderliche mechanische Stabilität des neuartigen Copolymers konnte unter den Extrembedingungen des Magenmilieus gezeigt werden. Eine vorzeitige Degradation des abbaubaren Polymers mit Wundheilungsstörungen konnte bei allen Versuchstieren ausgeschlossen werden. In weiterführenden Untersuchungen müssen die Mechanismen, die der Integration des Biomaterials in das umgebende Gewebe zugrunde liegen, sowie die Polymerdegradation und der Prozess des Geweberemodelings analysiert werden. Diese Erkenntnisse sind die Voraussetzung für eine hochspezifische Adaptation des neuartigen Polymers an die Bedingungen des jeweiligen Applikationsortes, um somit neuartige Therapieoptionen in der Medizin entwickeln zu können.

Reduced diagnostic value of lactate dehydrogenase (LDH) in the presence of radiographic contrast media.

Isoforms of the enzyme lactate dehydrogenase (LDH) were found in almost all cells of the organism and an elevated activity of LDH in the circulation is thought to be a clear indicator of elevated cell destruction coinciding with an increased release of components from the cellular cytoplasm, e.g. LDH. Here, we report on an in-vitro examination to test whether radiographic contrast media (RCM) could induce cell destruction followed by an increase in LDH release. The RCM were tested in non-flow cultures of human umbilical venous endothelial cells (HUVEC) of the fourth passage seeded on extracellular matrix and the results were compared to those from control cultures not exposed to contrast media. The examination revealed that the addition of contrast media to the cell culture media supplemented with pooled human serum (HSP) as source of exogenous LDH was followed by a strong decrease in LDH activity both in the absence and presence of HUVEC. Within 1.5 min after the addition of contrast media to the culture medium supplemented with HSP (30% vol of the culture medium were replaced by either of two contrast media, Iodixanol or Iopromide) the LDH activity decreased about 80% compared to the initial values. In contrast, the LDH activity did not change in cell culture media not supplemented with RCM. The partial replacement of HSP supplemented cell culture medium by RCM will cause a dilution of cell culture medium constituents. The decrease of LDH activity, however, was much stronger than the decrease thought to be attributable to the effects of dilution of cell culture medium, so that the role of dilution seems to be a minor one in this case. It has to be assumed that the RCM could interact with the LDH available in the culture medium as well as with the substrates delivered with the measurement system for the assessment of LDH activity, so that both, the amount of LDH and the activities of enzymes involved might be influenced. In the presence of HUVEC a similar effect was observed. Here, a little less strong decrease of LDH activity occurred compared to the decrease in cell culture medium without HUVEC. This was unexpected because a considerable amount of HUVEC were detached after the addition of contrast media and many of these cells were damaged seriously so that a significant amount of endogenous LDH should have been released. These unexpected results make it necessary to re-evaluate those past time examinations focussed on cell damage/destruction in the presence of contrast media, where the measurement of LDH activity was used as indicator or cell vitality and where cell decease rates were correlated to questionable toxic influences. According to the results of the examination reported here it is difficult to uphold the interpretation of recently published findings that contrast media almost exclusively induce cellular apoptosis and not necrosis.

Detaillierte Auswertung des Agarose-Diffusions-Tests im Rahmen der Biokompatibilitätsuntersuchung durch ein mikroskopisches Bildanalysesystem. Einfluß der Plasmasterilisation auf die Biokompatibilität eines neuartigen Photoset-Polymers / Detailed Evaluation of the Agarose Diffusion Test as a Standard Biocompatibility Procedure Using an Image Analysis System. Influence of plasma sterilization on the biocompatibility of a recently developed photoset-polymer

: In addition to sterilizability, biocompatibility is a further necessary property of biomaterials. The agarose diffusion test is an established standard in vitro procedure for investigating this property. The usual method of evaluating the agarose diffusion test, based on macroscopic determination of the size of the zone of decoloration, and microscopic assessment of cell lysis, limits the power of the test. To obtain more information on the impact of plasma sterilization on the biocompatibility of a new polymer, a high-resolution computer-aided morphometric image analysis system for the quantification of the agarose diffusion test at the cellular level has been developed. This was able to detect statistically significant differences in the cell lysis rate of plasma sterilized polymer specimens before and after 4 weeks of incubation in a physiological solution. The new method provides highly detailed information on the interaction of soluble material elements and cells. In addition to cell damage by potentially toxic elements, the sensitivity of the L929 fibroblasts used in the agarose diffusion test to mechanical stress also needs to be considered and verified by further investigations.

Results of Biocompatibility Testing of Novel, Multifunctional Polymeric Implant Materials In-Vitro and In-Vivo

Extensive in-vitro and in-vivo evaluation of the biocompatibility of biomaterials intended for clinical applications is necessary to provide information on the interactions that take place between the organism and these materials under specific implant conditions. Sterilization of polymer-based implant materials is a basic requirement but can lead to their damage or destruction. Low-temperature plasma (LTP) or ethylene oxide (EO) sterilization are objects of intensive research and were applied on the materials used in this program. After 4 weeks of polymer incubation in minimal essential medium (MEM), samples, sterilized by LTP, gave a markedly higher mean cell lysis rate (3.7 ± 2.5%) than EO sterilized samples (0.9 ± 0.3%). To achieve relevant in-vitro results on biomaterial-cell interactions, biocompatibility testing was carried out with cultures of locotypical cells, e.g. cells of the upper aerodigestive tract (ADT). Primary cell cultures of the oral cavity, the pharynx and the esophagus showed region-typical varying relationships between epithelial, fibroblastic and smooth muscle cells. Proper wound healing is thought to be required for the integration of biomaterials and angiogenesis is a prerequisite for this process. A focus of the present work was the influence of polymer-based implant materials and their degradation products on angiogenesis in-vivo. After 48 h, none of the polymer samples demonstrated development of an avascular region in the chorioallantoic membrane (CAM) test. A key process in proper wound healing is the tightly controlled degradation and regeneration of the extracellular matrix (ECM). A biomaterial for the reconstruction of the upper ADT is subjected to varying pH values and enzymatic, bacterial and mechanical stress during the digestive and the swallowing process. These complex conditions can currently only be investigated in-vivo in an animal model. As a model the stomach of the rat was selected, in which a biomaterial can be investigated under extreme chemical, enzymatic, bacteriological and mechanical conditions. Other parameters investigated are the impermeability of the polymer-tissue closure and the tissue regeneration after defect reconstruction. The fluid tight integration of a long term resorbable AB-copolymer network in the surrounding tissue of the gastric wall of Sprague Dawley rats could be demonstrated.