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Strategien zur Gewebsreparatur durch Zelltransplantate erfordern die Verfügbarkeit einer ausreichenden Menge von Zellen, die Schaffung konduktiver Mikrokulturbedingungen für die Integration und die Entwicklung des Implantats und die Entwicklung reproduzierbarer chirurgischer Technik für die klinische Anwendung des kultivierten Transplantats. In der vorliegenden Arbeit wurden mehrere Techniken der Zelltransplantation entwickelt und tierexperimentell erprobt. Unter Verwendung von Alginat wurde eine neue sequentielle Zellkulturtechnik für Knorpeltransplantate entwickelt. Der optimale Kompromiß zwischen der Matrixstabilisierung und einer ausreichenden Diffusionskapazität für die Zellfunktion wurde bei einer Mischung aus 0,6 % Alginat und 4,5 % Fibrin gefunden. Weitere untersuchte Matrixstrukturen zur Transplantation von Chondrozyten, wie die bioresorbierbaren Polymere, das Kollagen-Fibrin-Gel besitzen gegenüber der gegenwärtig kommerziell genutzten Methode hinsichtlich des chirurgischen Prozederes bei vergleichbaren histologischen Ergebnissen Vorteile. Die histomorphologischen Veränderungen und die Entwicklung des Transplantats in vivo werden durch die spezifischen Bedingungen der Transplantatumgebung beeinflußt. Dabei ist ein vollständiges zonales und sequentielles Remodeling von Knorpel-Knochendefekten nur bei nicht ausdifferenzierten Zellen (embryonale Chondrozyten, periostale Zellen) zu erkennen, da diese Zellen ein exzellentes chondrogenes und osteogenes Potential besitzen. Transplantate unter Verwendung von Chondrozyten zeigen dagegen nur eine sehr geringe Rekonstruktion des subchondralen Knochens. Periostale Zellen sind in vitro ohne Verlust des Phänotyps amplifizierbar und stellen daher eine optimale Zellquelle für das Tissue Engineering dar. Für das Bone Engineering ist die Kombination der osteokonduktiven Eigenschaften unterschiedlicher Trägermaterialien mit Zellen, die ein osteogenes Potential besitzen ein neuer Weg zur Optimierung des Prozesses der knöchernen Rekonstruktion, wie in Versuchen zur Rekonstruktion segementaler Ulnadefekte bei Kaninchen gezeigt werden konnte. Die Herstellung eines präossären stabilen aber formbaren Transplantats mit vielfältigen klinischen Einsatzmöglichkeiten ist unter Verwendung von biodegradierbaren Polymeren und von Fibrinbeads realisierbar. Der Einsatz von Wachstumsfaktoren, wie TGF-?1 und die zunehmenden Erkenntnisse zu den Zell-Zell- und Zell-Matrix-Interaktionen ermöglichen die verbesserte Generation ortsständigen Gewebes durch multipotente Zellen. Die immer komplexere und umfassendere Wiederholung der sich in der Ontogenese abspielenden Vorgänge durch die Techniken des Tissue Engineering, ermöglicht die Schaffung therapeutischer Optionen zur Behandlung von Knochen- und Knorpeldefekten, wo bisher keine existierten oder nur unzulänglich vorhanden waren. / Strategies for tissue repair by cell transplants require the availability of a sufficient amount of cells, the creation of conductive microculture conditions for the integration and development of the implant and the development of reproducible surgical techniques for the clinical application of the cultivated transplant. Within the frame of the present work, several techniques of cell transplantation were developed and tested by way of experiment. By using alginate, a new sequential cell culture technique was developed for cartilage transplants. The optimum compromise between the matrix stabilization and a sufficient diffusion capacity for the cell function was found with a mixture of 0.6 % of alginate and 4.5 % of fibrin. Further investigated matrix structures for the transplantation of chondrocytes, such as the bio-absorbable polymers, the collagen fibrin jelly show advantages compared with the method commercially applied at present regarding the surgical procedure with the gained histological results being comparable. The histomorphological changes and the development of the transplant within the living body are influenced by the specific conditions of the transplant environment. In this connection, a complete zonal and sequential remodeling of osteochondrodefects can only be detected for non-outdifferentiated cells (embryonic chondrocytes, periosteal cells) as these cells have an excellent chondrogenic and osteogenic potential. When using chondrocytes for transplants, however, the transplant only shows a very little restoration of the subchondral bone. Periosteal cells can be amplified in the living body without losing the phenotype, thus constituting an optimum cell source for tissue engineering. For the bone engineering, the combination of the osteoconductive properties of different carrier materials with cells having an osteogenic potential is a new way for optimizing the process of bone restoration as it was demonstrated in tests for the restoration of segmental ulnar defects occurring with rabbits. The generation of a preosteal stable, but mouldable transplant with manifold clinical possibilities of utilization can be realized by using biodegradable polymers and fibrin beads. The use of growth factors, such as TGF-?1, and the increasing knowledge of cell-cell and cell-matrix interactions enable the improved generation of stationary tissue by multipotent cells. The more and more complex and comprehensive repetition of processes going on in the ontogenesis by way of tissue engineering enables the creation of therapeutic options for the treatment of osteochondrodefects where hitherto none existed or just in a too small number.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Autologous bone grafting is commonly used throughout the world. Autograft both fills the void and is bone inductive, housing the particular cells that are needed for bone regeneration. However, a regenerative complement to autograft is of great interest as the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of a limited volume of material. Two such regenerative products that utilise bone morphogenetic protein (BMP)-7 and -2 have been used for more than a decade clinically. Unfortunately, several side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery and use of supra physiological doses of the BMPs due to poor localisation and retention of the growth factor. The purpose of this thesis was to harness the strong inductive capacity of the BMP-2 by optimising the carrier of this bioactive protein, thereby minimising the side effects that are associated with the clinical products and facilitating safe and localised bone regeneration. We focused on an injectable hyaluronan-based carrier developed through polymer chemistry at the University of Uppsala. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident enzymes. Earlier studies have shown improved properties when adding hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that bone formation was enhanced when using nano-sized hydroxyapatite. In Paper II, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. We wished to further develop the carrier system, but were lacking an animal model with relatively high throughput, facilitated access, paired data, and we were also committed to the 3Rs of refinement, reduction, and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper III. In Paper IV, we sought to further optimise the biomaterial properties of the hydrogel through covalent bonding of bisphosphonates to the hyaluronan hydrogel. This resulted in exceptional retention of the growth factor BMP-2. In Paper V, SPECT/PET/µCT was combined as a tri-modal imaging method to allow visualisation of the biomaterial’s in situ action, in terms of drug retention, osteoblast activity and mineralisation. Finally, in Paper VI the correlation between existing in vitro results with in vivo outcomes was observed for an array of biomaterials. The study identified a surprisingly poor correlation between in vitro and in vivo assessment of biomaterials for osteogenesis.

Bone is a remarkable multifunctional tissue with the ability to regenerate and remodel without generating any scar tissue. However, bone loss due to injury or diseases can be a great challenge and affect the patient significantly. Transplanting bone graft from one site in the patient to the site of fracture or bone void, i.e. autologous bone grafting is commonly used throughout the world. The transplanted bone not only fills voids, but is also bone inductive, housing the particular cells that are needed for bone regeneration. Nevertheless, a regenerative complement to autograft is of great interest and importance because the benefits from an off-the-shelf product with as good of healing capacity as autograft will circumvent most of the drawbacks with autograft. With a regenerative-medicine approach, the use of biomaterials loaded with bioactive molecules can avoid donor site morbidity and the problem of limited volume of material. Two such regenerative products that utilize bone morphogenetic protein 7 and 2 have been used for more than a decade in the clinic. However, some severe side effects have been reported, such as severe swelling due to inflammation and ectopic bone formation. Additionally, the products require open surgery, use of supra physiological doses of the BMPs due to poor localization and retention of the growth factors. The purpose of this thesis was to harness the strong inductive capability of the BMP-2 by optimizing the carrier of this bioactive protein, thereby minimizing the side effects that are associated with the clinical products and facilitating safe and localized bone regeneration at the desired site. We focused on an injectable hyaluronan-based carrier. The strategy was to use the body’s own regenerative pathway to stimulate and enhance bone healing in a manner similar to the natural bone-healing process. The hyaluronan-based carrier has a similar composition to the natural extracellular matrix and is degraded by resident hyaluronidase enzymes. Earlier studies have shown a more controlled release and improved mechanical properties when adding a weight of 25 percent of hydroxyapatite, a calcium phosphate that constitutes the inorganic part of the bone matrix. In Paper I, the aim was to improve the carrier by adding other forms of calcium phosphate. The results indicated that the bone formation was enhanced when using nano-sized hydroxyapatite. We wished to further develop the carrier system but were lacking an animal model with high output and easy access. We also wanted to provide paired data and were committed to the 3 Rs of refinement, reduction and replacement. To meet these challenges, we developed and refined an animal model, and this is described in Paper II. In Paper III, we characterized and optimized the handling properties of the carrier. In Paper IV, we discovered the importance of crushing the material, thus enhancing permeability and enlarging the surface area. In Paper V, we sought to further optimize biomaterial properties of the hydrogel through covalently bonding of bisphosphonates to the hyaluronan hydrogel. The results demonstrated exceptional retention of the growth factor BMP-2. In Paper VI, the in vivo response related to the release of the growth factor was examined by combining a SPECT/PET/µCT imaging method to visualize both the retention of the drug, and the in-vivo response in terms of mineralization.