Andreas Haisch

Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction.

OBJECTIVE An important role in joint and cartilage homeostasis in adults has been demonstrated recently for morphogenetic factors of the transforming growth factor beta family. Therefore, this study was undertaken to investigate the potential of bone morphogenetic proteins (BMPs) in chondrocyte differentiation using current technologies of tissue engineering. METHODS Complementary DNAs of recombinant human BMPs 2, 4, 5, 6, and 7 were transfected into primary bovine articular chondrocytes. Transgenic chondrocytes were assembled 3-dimensionally in alginate or in bioresorbable co-polymer fleeces of vicryl and polydioxanon embedded in low-melting-point agarose. Redifferentiation and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice were assayed by semiquantitative reverse transcriptase-polymerase chain reaction, histology, and in situ hybridization, and findings were compared with those in unmodified or control-transfected primary chondrocytes. RESULTS Compared with other BMPs and control vector, BMP-7 induced a decrease in type I collagen expression in artificial cartilage, while transcription of the cartilage-specific type II collagen remained stable. In transplantation experiments, BMP-7 transgenic cartilage revealed the greatest amount of matrix synthesis, and BMP-7 was the only morphogen to suppress the infiltrative response of mouse fibroblastic cells into engineered cartilage, thereby preventing transplant destruction. CONCLUSION Cartilage differentiation and matrix maturation are promoted by BMPs in cartilage engineering. The inhibitory effect of BMP-7 on a nonspecific infiltrative response in immunocompromised nude mice further suggests that individual morphogens not only may contribute to cartilage maturation, but also may protect it from nonspecific inflammation and invasive destruction. These properties advance BMPs as promising tools for engineering of cartilaginous joint bioprostheses and as candidate biologic agents or genes for cartilage stabilization in arthritis.

A tissue-engineering model for the manufacture of auricular-shaped cartilage implants.

Abstract. The established surgical methods of external ear reconstruction using autogenous tissue represent the current state of the art. Because of the limited possibilities for shaping conventional harvested autogenous rib cartilage, the cosmetic results of auricular reconstruction are frequently unsatisfactory. Tissue engineering could represent an alternative technique for obtaining a precisely shaped cartilage implant that avoids donor site morbidity and unsatisfactory cosmetic results. In this study, the reliability and quality of a tissue-engineering model for the manufacture of auricular-shaped human cartilage implants was investigated, focusing on the feasibility of the manufacturing process and the in vivo and in vitro maturation of an extracellular cartilage-like matrix. Implants were molded within an auricular-shaped silicone cylinder, and human nasal septal chondrocytes crosslinked by human fibrin within bioresorbable PGLA-PLLA polymer scaffolds were used. After an in vitro incubation of up to 6 weeks, defined fragments of the prefabricated auricular-shaped construct were implanted subcutaneously on the backs of nude mice for at least 6 to 12 weeks (n=7). Scaffolds without cell loading served as controls. Macroscopic and histochemical examination after 3 and 6 weeks in vito showed a solid compound of homogenously distributed chondrocytes within the polymer scaffold, leading only to a limited pericellular matrix formation. Analysis after 6 and 12 weeks of in vivo maturation demonstrated a solid tissue compound and neocartilage formation with the presence of cartilage-specific matrix components. Implants obtained shape and size during the entire period of implantation. The model of cartilage implant manufacturing presented here meets all biocompatible requirements for in vitro prefabrication and in vivo maturation of autogenous, individually shaped cartilage transplants.

Mechanical quality of tissue engineered cartilage: results after 6 and 12 weeks in vivo.

Traumatic events are a primary cause for local lesions of articular cartilage. If treated early, restoration of the initial joint geometry and integrity may be achieved. In large defects, sufficient material is not available to bridge the affected area. Heterologeous transplantation is not well accepted due to the risk of infection and immune response. Alternatives are cartilage-like structures, which may be cultured in vitro and transplanted into the defect site. Critical to the success of these new tissues are their mechanical properties. Goals of this study were to generate a hyaline-like cartilage structure, to evaluate its performance in vivo and to verify that its cellular and material properties meet those of native cartilage. Hyaline-like cartilage specimens were generated in vitro and implanted in the backs of nude mice. Specimens were explanted after 6 and 12 weeks, mechanically tested using an indentation test and histologically examined. In mechanical testing, stiffness and failure load significantly increased between weeks 6 and 12. At 12 weeks, mechanical properties of the hyaline-like cartilage were comparable to those of native nasal septal cartilage. Compared to native articular cartilage, the engineered tissue achieved up to 30-50% in strength and mechanical stiffness. In histological examination, specimens showed neocartilage formation. The mechanical testing procedure proved to be sufficiently sensitive to identify differences in properties between cartilage specimens of different origin and at different stages of healing. As an adjunct to histological analysis, mechanical testing may be a valuable tool for judging the utility of engineered cartilage prior to a broad clinical usage.

Human platelet supernatant promotes proliferation but not differentiation of articular chondrocytes

The objective of the study was to evaluate the growth-promoting activity of human platelet supernanant on primary chondrocytes in comparison with fetal calf serum (FCS) supplemented cell culture medium. Furthermore, the differentiation potential of platelet supernatant was determined in three-dimensional artificial cartilage tissues of bovine articular chondrocytes. Proliferation of articular and nasal septal chondrocytes was assayed by incorporation of BrdU upon stimulation with ten different batches of human platelet supernatant. On bovine articular chondrocytes, all these batches were at least as growth-promoting as FCS. On nasal septal chondrocytes, nine out of ten batches revealed increased or equivalent mitogenic stimulation compared with medium supplemented with FCS. Three-dimensional culture and subsequent histological analysis of matrix formation were used to determine the differentiation properties of platelet supernatant on articular chondrocytes. Human platelet supernatant failed to induce the deposition of typical cartilage matrix components, whereas differentiation and matrix formation were apparent upon cultivation of articular chondrocytes with FCS. Proliferation assays demonstrated that human platelet supernatant stimulates growth of articular and nasal septal chondrocytes; however, platelet supernatant failed to stimulate articular chondrocytes to redifferentiate in three-dimensional chondrocyte cultures. Therefore platelet lysate may be suitable for chondrocyte expansion, but not for maturation of tissue-engineered cartilage.

Tissue-engineering humanen Knorpelgewebes für die rekonstruktive Chirurgie unter Verwendung biokompatibler resorbierbarer Fibringel- und Polymervliesstrukturen

ZusammenfassungMittels des noch jungen Forschungsgebietes Tissue engineering wird erstmals in-vitro hergestelltes lebendes Ersatzgewebe für den Einsatz in Klinik und Forschung verfügbar. Die bisher verwendeten In-vitro-Modelle zur Züchtung von Knorpelgewebe lassen eine klinische Anwendung aufgrund fehlender Daten zur Biokompatibilität und fehlender plastischer Stabilität nicht zu. Das in dieser Untersuchung vorgestellte Modell verwendet 2 biokompatible Materialien, die sich seit Jahren in verschiedenen Bereichen der operativen Medizin in klinischer Anwendung befinden. Zur dreidimensionalen Verteilung der Zellen innerhalb eines resorbierbaren Polyglycolid-Polylactid-Copolymers werden die Zellen innerhalb der Polymerstruktur mittels Fibrin vernetzt. Anhand von histo- und zellmorphologischer Untersuchungsmethoden konnte gezeigt werden, daß das vorgestellte Modell optimale Voraussetzungen zur Herstellung von Knorpelgewebe bietet. Immunhistochemisch konnten knorpelspezifische Bestandteile wie Kollagen II, Chondroitinsulfat und Knorpelproteoglycan nachgewiesen werden. Histomorphologisch ließ sich ein über mindestens 5 Wochen formstabiler Zellverband darstellen. Da die Fibrinvernetzung unter ausschließlicher Verwendung autologer Komponenten durchgeführt werden kann, bietet dieses Modell erstmals die Grundlage zur In-vitro-Herstellung eines biokompatiblen, autologen Knorpelgewebes für die plastisch-rekonstruktive Chirurgie.SummaryCurrent practical approaches in cartilage engineering still face problems with threedimensional cell distribution or require components for cell immobilization, raising biocompatibility problems. In this study, we present a new model using cells cross-linked by fibrin within biocompatible resorbable polymers. Both components have been in clinical use for a long time. Immunohistochemical procedures showed that this model provides optimal requirements for in vitro cartilage production. Immunochemically, cartilage-specific extracellular components such as proteoglycan, chondroitin sulfate and collagen II were characterized. Histomorphological methods showed a mechanically stable tissue compound that lasted for at least 5 weeks. This model may be the first to provide all biocompatible requirements for in vitro production of autologous cartilage transplants for reconstructive surgery.

Macroencapsulation of human cartilage implants: pilot study with polyelectrolyte complex membrane encapsulation.

Autogenous cartilage transplantation is a generally accepted method in reconstructive surgery. A promising alternative to this established method could be represented by in vitro engineering of cartilage tissue. In both methods of autogenous transplantation, host response induces reduction of transplant size and transplant instability to an unforeseeable extent. To investigate if polyelectrolyte complex (PEC) membranes were able to avoid host-induced effects on implanted tissues without neglecting the tissue metabolism, human septal cartilage was encapsulated with polyelectrolyte complex membranes and subcutaneously implanted on the back of nude mice. Septal cartilage implants, without encapsulation served as control group. Histochemical and electron microscopic investigations were performed 1, 4, 8 and 16 weeks after implantation. In the case of an intact PEC-membrane no interactions between the host and the implant could be observed. In some implants, the capsule was torn in several areas and signs of chronic inflammation with the cartilage having been affected mildly could be observed. Implanted cartilage protected with PEC-encapsulation showed no signs of degeneration and significantly lower level of after effects of chronic inflammation than implanted cartilage without PEC-encapsulation. Therefore, it could be expected, that PEC membrane encapsulation offers a novel approach to protect cartilage implants from host response after autogenous transplantation.

Bilateral synchronous tonsillar carcinoma in cervical cancer of unknown primary site (CUPS)

In addition to panendoscopy and imaging for staging, ipsilateral tonsillectomy is a standard procedure in the search for the primary tumor in cervical cancer of unknown primary site (CUPS). It is not clear from the literature, whether bilateral tonsillectomy has been established as the standard procedure in cancer of unknown primary origin. A bilateral synchronous tonsillar carcinoma has thus far only been described three times in the literature. We report on a case of CUPS in which a bilateral tonsillar carcinoma was detected after bilateral tonsillectomy. We also discuss the inclusion of bilateral tonsillectomy as a standard procedure in the search for primary malignancies. To diagnose and adequately treat a bilateral synchronous tonsillar carcinoma without losing time, we recommend bilateral tonsillectomy as a standard procedure in cervical CUPS.

Resorptionsprotektion autogener Knorpeltransplantate durch Polyelektrolytkomplexmembranverkapselung

ZusammenfassungIn der rekonstruktiven Chirurgie könnte neben der etablierten Methode der autogenen Knorpeltransplantation das Tissue engineering humaner Knorpelgewebe zunehmend an Bedeutung gewinnen. Resorptive Mechanismen können jedoch bei beiden Methoden zu funktionell und kosmetisch unbefriedigenden Ergebnissen führen. Unter den heute zur Verfügung stehenden Verfahren der Gewebeprotektion könnte die Polyelektrolytmembranverkapselung mit Natriumzellulosesulfat und Polydialylldimethylammoniumchlorid einen ausreichenden Schutz vor resorptiven Einflüssen gewährleisten. Humaner nativer Nasenseptumknorpel wurde verkapselt (Gruppe 1) und unverkapselt (Gruppe 2) subkutan in thymusaplastische Nacktmäuse implantiert. Die Transplantate wurden nach 1, 4, 8, 12 und 16 Wochen explantiert und histologisch sowie rasterelektronenmikroskopisch untersucht. Gruppe 1 zeigte über den gesamten Implantationszeitraum keinerlei Resorptionsmerkmale bei vollständig erhaltener Knorpelgrundsubstanz. Gruppe 2 hingegen zeigte, korrelierend mit der Implantationsdauer, in zunehmendem Maße Knorpelzellnekrosen und einen bindegewebigen Umbau im Sinne einer Resorption. Die Polyelektrolytmembranverkapselung könnte somit eine sichere Methode zum Schutz humaner Knorpeltransplantate vor resorptiven Einflüssen darstellen. Für den Plastisch-rekonstruktiven Chirurgen wird hierdurch das gewünschte kosmetische und funktionelle Ergebnis kalkulierbarer.SummaryIn reconstruction of cartilage defects, autogenous transplantation is known as a reliable and experienced method. Although a clinical application has not been reported until now, tissue engineering permits in vitro production of autogenous cartilage transplants. Nevertheless, in both methods the cartilage is exposed to individually varying resorptive mechanisms. Among other methods for in vivo tissue protection, the encapsulation with a semipermeable polyelectrolytecomplex membrane could guarantee sufficient protection against resorptive influences. Human septal cartilage was encapsulated (group 1) with polyelectrolytecomplex membranes and subcutaneously implanted on the back of thymusaplastic nude mice. Cartilage implants without encapsulation (group 2) were used as control. Scanning electron microscopy and histochemical investigations were performed 1, 4, 8, 12 and 16 weeks after implantation. Group 1 showed no signs of resorption and chronic inflammation at all. In contrast, group 2 presented, correlating to the time of implanta-tion, increasing signs of cell death and fibrotic transformation, representing an increased activity of resorption. In conclusion, tissue encapsulation with a polyelectrolytecomplex membrane could ensure a sufficient protection of human cartilage transplants from resorptive influences. For the plastic-reconstructive surgeon the desired result becomes more calculable.

Das myoepitheliale Karzinom (malignes Myoepitheliom) der Speicheldrüsen Eine Falldarstellung

ZusammenfassungEs wird der seltene Verlauf der Entstehung eines myoepithelialen Karzinoms in einem pleomorphen Adenom der kleinen Speicheldrüsen der Mundschleimhaut vorgestellt. Der im linken Mundwinkel angesiedelte Tumor zeigte primär ein dominierendes pleomorphes Adenom der kleinen Speicheldrüsen mit einem kleinen kapselnahen Areal eines myoepithelialen Karzinoms. Im Verlauf von 2 und 5 Jahren entwickelte sich im Bereich des linken Mundwinkels 2malig ein erbsengroßer Rezidivtumor. Die Tumoren wurden großzügig exzidiert. Die histologische Aufarbeitung zeigte jeweils ein myoepitheliales Karzinom der kleinen Speicheldrüsen, Anteile eines pleomorphen Adenoms ließen sich nicht mehr nachweisen. Die Klinik, Diagnostik, Therapie und histologischen Merkmale unter Berücksichtigung der Literatur werden diskutiert.SummaryWe report the rare development of a myoepithelial carcinoma in a pleomorphic adenoma involving the minor salivary glands in the buccal mucosa of a 78-year-old female. Tumor presented as a small asymptomatic left buccal mass. The initial dominant component of the neoplasm was a pleomorphic adenoma, while the minor component was a myoepithelial carcinoma. Recurrences of tumor after 2 and then 5 years were excised. Histopathological examination of the last tissue removed showed a pure myoepithelial carcinoma of the minor salivary glands without evidence for a pleomorphic adenoma. The clinical features, therapy, diagnosis, histopathology and literature are reviewed.