Olaf Schultz

Segmental bone repair by tissue-engineered periosteal cell transplants with bioresorbable fleece and fibrin scaffolds in rabbits

The biological bone healing depends on the presence of osteochondral progenitors and their ability for proliferation. Isolated periosteal cells were seeded into biodegradable PGLA polymer fleece or fibrin beads and cultivated for 14 days after prior monolayer culture. On 12 New Zealand white rabbits 8 mm metadiaphyseal ulna defects were created bilaterally and subsequently filled with cell-fibrin beads, with polymers seeded with cells compared to controls with fibrin beads and polymers alone and untreated defects. A semiquantitative grading score was applied for histomorphological and radiological analysis after 28 days. Histologically intense bone formation was observed in both experimental groups with cell transplants only. The histological and radiological scoring was superior for both experimental groups. Control groups revealed only poor healing indices and untreated defects did not heal. The highest histological score was noted in the group with polymer fleeces containing periosteal cells. Applying the radiographic score system we determined a significant difference between experimental groups and controls without cells. The radiographic and histological scores for both experimental groups containing periosteal cells differed not significantly. The results strongly encourage the approach of the transplantation of pluripotent mesenchymal cells within a suitable carrier structure for the reconstruction of critical size bone defects.

Porcine mesenchymal stem cells

Abstract. The potential of mesenchymal stem and progenitor cells (MSC) to replicate undifferentiated and to mature into distinct mesenchymal tissues suggests these cells as an attractive source for tissue engineering. The objective was to establish a protocol for the isolation of porcine MSC from bone marrow and to demonstrate their ex vivo differentiation into various mesenchymal tissue cells. MSC from passage 2 were selected for differentiation analysis. Differentiation along the osteogenic lineage was documented by deposition of calcium, visualization of alkaline phosphatase activity, and by analysis of osteogenic marker genes. Adipocytes were identified morphologically and by gene-expression analysis. Deposition of type II collagen and histological staining of proteoglycan indicated chondrogenic differentiation. Therefore, porcine MSC may be introduced as a valuable model system with which to study the mesenchymal lineages for basic research and tissue engineering.

Artificial tissues in perfusion culture.

In the stagnant environment of traditional culture dishes it is difficult to generate long term experiments or artificial tissues from human cells. For this reason a perfusion culture system with a stable supply of nutrients was developed. Human chondrocytes were seeded three-dimensionally in resorbable polymer fleeces. The cell-polymer tissues were then mounted in newly developed containers (W. W. Minuth et al, Biotechniques, 1996) and continuously perfused by fresh medium for 40 days. Samples from the effluate were analyzed daily, and the pH of the medium and glucose concentration remained stable during this period. The lactid acid concentration increased from 0,17 mg/ml to 0,35 mg/ml, which was influenced by the degradation of the resorbable polymer fibers used as three dimensional support material for the cells. This perfusion system proved to be reliable especially in long term cultures. Any components in the culture medium of the cells could be monitored without disturbances as caused by manual medium replacement. These results suggest the described perfusion culture system to be a valuable and convenient tool for many applications in tissue engineering, especially in the generation of artificial connective tissue.

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.

The influence of transforming growth factor β1 on mesenchymal cell repair of full-thickness cartilage defects

To repair full-thickness articular cartilage defects in rabbit knees, we transplanted periosteal cells in a fibrin gel and determined the influence of transforming growth factor beta (TGF-beta) in vitro. Alginate served as a temporary supportive matrix component and was removed prior to transplantation. The defects were analyzed macroscopically, histologically, and electron microscopically, and evaluated with a semi-quantitative score system. Periosteal cell transplants showed a chondrogenic differentiation, which results in the development of embryonic-like cartilage tissue after 4 weeks and complete resurfacing of the patellar groove after 12 weeks. In the control groups, no repair was observed. Under the influence of TGF-beta1 we observed a reduction of the cartilage layer, whereas the osteochondral integration and the zonal architecture were improved. Periosteal cell-beads are stable cartilage transplants and have stiffness and elasticity enough for easy and sufficient transplant fixation. Further investigations are necessary to optimize the application of TGF-beta1 for cartilage repair.

Tissue engineered cartilage repair using cryopreserved and noncryopreserved chondrocytes.

The objective of this study was to reconstruct full thickness cartilage defects in rabbit knees with in vitro engineered cartilage tissue based on noncryopreserved and cryopreserved chondrocytes in polymer fleece scaffolds. Osteochondral defects in rabbits were filled with polymer cylinders with noncryopreserved or cryopreserved allogeneic chondrocytes and compared with empty defects and defects filled with polymers alone. The defects were evaluated macroscopically and histologically 4 and 12 weeks after surgery. Transplant samples were graded using a semiquantitative score system. Successful healing was defined as complete integration of a hyalinelike and structurally intact cartilage into the defect and occurred in 71% of the group with noncryopreserved chondrocytes after 4 weeks and 100% of the rabbit knees after 12 weeks, whereas hyalinelike cartilage was seen in 71% of the group with cryopreserved chondrocytes after 4 weeks, and in 85% after 12 weeks. No newly formed cancellous bone was present in the subchondral bone. In the control groups, no cartilagelike tissue was seen. Transplantation of chondrocytes in polymer fleece constructs is a suitable approach for joint cartilage repair. Noncryopreserved chondrocytes are preferred to cryopreserved chondrocytes because of their regenerative potential. In vitro engineered cartilage offers broad opportunities for optimization of cartilage transplantation based on the controlled use of morphogenic and biologically active factors such as transforming growth factor-beta and bone morphogenetic proteins.

Emerging strategies of bone and joint repair

The advances in biomedicine over the past decade have provided revolutionary insights into molecules that mediate cell proliferation and differentiation. Findings on the complex interplay of cells, growth factors, matrix molecules and cell adhesion molecules in the process of tissue patterning have vitalized the revolutionary approach of bioregenerative medicine and tissue engineering. Here we review the impact of recent work in this interdisciplinary field on the treatment of musculoskeletal disorders. This novel concept combines the transplantation of pluripotent stem cells, and the use of specifically tailored biomaterials, arrays of bioactive molecules and gene transfer technologies to direct the regeneration of pathologically altered musculoskeletal tissues.

Chondrozytentransplantation in PGLA/Polydioxanon-Vliesen

ZusammenfassungDie Transplantation von chondrogenen Zellen in supportiven Trägerstrukturen erweist sich zunehmend als eine alternative Methode zur Behandlung von Knorpeldefekten. Gegenstand der vorliegenden Untersuchung war die Transplantation allogener Chondrozyten in einem biodegradierbaren Polymervlies (PGLA/Polydioxanon) in Gelenkknorpeldefekte in einem Kaninchenmodell. In Knorpel-Knochen-Defekte der femoralen Patellagleitbahn wurden Transplantate aus isolierten allogenen Chondrozyten in einem bioresorbierbaren Polymerkonstrukt eingesetzt und mit Leerdefekten sowie Polymerkonstrukten ohne Zellen als Kontrollgruppen verglichen. Die Resultate wurden nach 4 und 12 Wochen histologisch und histochemisch beurteilt. Die Beurteilung der Transplantatintegration und der Architektur des neugebildeten Knorpels erfolgte mit einem semiquantitativen Score. Bereits nach 4 Wochen war die Entwicklung der Zell-Polymer-Transplantate zu einem hyalinartigen Knorpel nachweisbar, nach 12 Wochen waren die Defekte fast vollständig mit hyalinartigem Knorpel gefüllt. Veränderungen der Transplantatstruktur durch die Biodegradation des Trägermaterials wurden nicht beobachtet. Die Defekte der Kontrollgruppen wiesen keine Heilungszeichen auf oder heilten mit Faserknorpel. Die durchgeführten Untersuchungen zeigten, dass sich resorbierbare Polymere aufgrund der Möglichkeit der In-¶vitro-Generation eines semisoliden Knorpeltransplantats und der resultierenden einfachen Fixationsmöglichkeit im Defekt als geeignetes Trägermaterial für die Knorpeltransplantation erwiesen. In vivo stellen sie ein adäquates Mikromilieu für die Bildung von hyalinem Knorpel dar und lassen bei Bindung von Wachstumsfaktoren durch eine protrahierte Freisetzung eine weitere Verbesserung der Ergebnisse nach Chondrozytentransplantation erwarten.SummaryThe transplantation of chondrogenic cells in a supportive carrier structure proved to be a promising alternative for the treatment of cartilage defects. In the study presented we focused on the transplantation of allogeneic chondrocytes in a biodegradable polymer scaffold (PGLA/Polydioxanon) in articular cartilage defects in a rabbit defect model. Isolated allogeneic chondrocytes embedded in a PGLA polymer scaffold were transplanted into osteochondrogenic defects of the patellar groove and compared with empty defects and transplants of polymer scaffolds without cells. The histological and histochemical analysis was performed after 4 and 12 weeks. The transplant integration and the architecture of the newly formed cartilage were evaluated with a semiquantitative score. After 4 weeks the development of a hyaline-like cartilage tissue of the cell-polymer-transplants was observed, after 12 weeks the defects were nearly completely filled with hyaline-like cartilage. The biodegradation of the polymer construct did not affect the histological structure of the transplant area. Defects of the groups with empty defect and polymer transplants without cells revealed no or insufficient healing indices. The study demonstrated that biodegradable polymers served as suitable carriers for the chondrocyte transplantation, which is due to the in-vitro establishment of a semi-solid cartilage transplant and the resulting effective transplant fixation into the defect. In-vivo the polymer cell transplants seem to provide a supportive microenvironment for the development of hyaline cartilage. The controlled release of morphogenic factors or bioactive molecules and the use of pluripotent mesenchymal progenitor cells opens new perspectives for the optimization of cartilage repair procedures.