In vitro development of a ligament and in vivo analysis of the bone-soft tissue interface

Ligament injuries frequently appears in the young active part of our population. Ligaments are important structures as they provide stability to the joints. Injuries to the ligaments may cause instability in the injured joint which commonly leads to improper function and pain. A common example of such injuries are the anterior cruciate ligament injuries, which commonly appears in pivoting sports, such as european handball, soccer and alpine skiing. The healing potential of ligament injuries are limited, thus surgical reconstruction is a commonly performed orthopedic procedure aiming to restore the stability of the joint to relieve the patients pain and regain function. This kind of surgery is both technically demanding and depending a fragile biological process of a tendon graft healing in a bone tunnel, and the long term results are limited. The aim of this project is to unveil new and better treatment options for this group of patients. We aim to investigate cell signaling pathways involved in regulating cell differentiation and deposition of extracellular matrix to improve the outcome for ligament injuries. This by investigating the in vitro, ex vivo and in vivo effect of different growth factors and different drug vehicles to enhance the ligamentization of tendon grafts used for reconstructions. We aim to improve current knowledge about fibroblast differentiation in tendon grafts, possibly to favorably altering of the extracellular content and organization for improved biomechanical properties and enhanced osseointegration in the adjacent bones to improve the outcome for these patients. A device was developed to characterize tissue cultured under different biological environment, both regarding tension and growthfactors. This culturing system has shown promising results for individualized analysis of tissue cultured under tension to mimic their natural environment, as tendons, ligament, and pathologic fibrotic tissue from Dupuytrens disease. This system will facilitate further studies to elucidate cellular pathways involved in cellular hemostasis, extra cellular matrix deposition and disease progression. Further, we investigated the in vivo the effect of selected bone anabolic growthfactors (BMP" and GSK126) to enhance the osseointegrating of tendons healing in bone tunnels. These selected agents have proven to be powerful stimulator of osteoblast differentiation and new bone formation, thus have potential to enhance the healing and anchoring of a traditional tendon graft in a bone tunnel, thus opening for a new clinical application. It is important to increase the strength of the reconstruction, to promote allow early rehabilitation for the patients and to provide more reliable long term clinical outcome. A verified animal model was used for this experiment. We found tendency of increased biomechanical properties in bone anabolic treated reconstructions, compared to saline. In addition, BMP2 seemed to increase the formation of bone in the bone tunnel, however not significant. Histological analysis did not unveil any obvious differences between the groups. As such, local administration of bone anabolic factors may enhance the incorporation of a tendon graft in a bone tunnel, however our findings must be verified in additional experiments and clinical trails before incorporated in the clinical management. In addition, we wanted to assess the effect of an articial graft, as an enhancement of a tendon graft or on its own, used in a ligament reconstruction. This to allow for early active rehabilitation and possible quicker return to sport activities, but also to assess the capability of the graft to serve as a scaffold for tissue regeneration. A verified animal modell on anterior cruciate ligament reconstruction was used. Artificial graft alone, in combination with a tendon graft, was compared to tendon graft alone. Our biomechancial test revealed improved capabilites in the animals with artificial grafts compared to regular autograft. There were no difference in new bone formation in the bone tunnels, and we did not find any difference in histological analysis og gene expression analysis indication the artificial graft induce inflammation or other adverse effect within the joint. We did not find any regeneration of ligament like tissue along the artificial graft. Thus, the artificial graft seems to enhance the biomechancial properties of a ligament reconstruction, that may facilitate earlier and more active rehabilitation. No adverse effects was detected indicating the artificial graft is not safe to use. Therefor the artificial graft seems to be capable to protect a ligament reconstruction in the early fragile phase, however we did not find convincing signs of tissue regeneration along the graft. Our findings must be confirmed in further studies.

Utvikling av en inkubator for å analysere effekt av tensjon og miljø på vev i dyrkning. Bedret forståelse av benstimulerende midlers effekt på sene ben tildeling. Bedre forståelse for bruk av kunstig forsterkning av sener brukt som graft i leddbåndsrekonstruksjon. PhD kandidaten har vært involvert i betydelig grad i ialt 7 vitenskaplige publikasjoner i internasjonale tidsskrift i perioden, og ytterligere 6-7 vitenskaplige artikler er under fremstilling og/eller sendt inn til vurdering for publikasjon. Det er etablert kontakt og samarbeid med internasjonal forskningsgruppe ved Mayo Clinic, ROchester, USA. Kompetansehevning ved bruk av dyremodeller og læring av ny metoder, inkludert biomolekylære analyser som qPCR. PhD kandidat har gjennomført forskningsutdanning ved Universitetet i Oslo, og utenlandsopphold ved institusjon i USA.

Intra-articular ligament injuries are increasing in incidence. The primary healing potential of these injuries are poor with their intrasynovial location. Untreated they can lead to abnormal articular loading and progressive degenerative changes of the joint, causing pain and loss of function for the patient. Surgical reconstruction often does not relieve the patient?s pain and joint instability. To improve long term outcome for these patients new treatment options is required. This study aims to bridge the gap between tissue engineering and cellular based therapies with bone marrow stromal cells (BMSC). The primary goal of this project is to develop a neoligament in vitro using polymer scaffolds seeded with BMSC and enhance the bone-soft tissue interface through selective differentiation induction. We will evaluate segmental BMSC differentiation along synthetic and allograft scaffolds, to find a scaffold that will enhance both cellular growth, as well as cellular differentiation. This neoligament will be laden with BMSCs and growth factors to enhance osseointegration at the fibrocartilaginous bone-soft tissue interface, while generating native ligamentous tensile properties throughout the body of the ligament. We will evaluate in vivo the effect of biological treatment on the bone-soft tissue interface. A verified animal experimental model will be used to assess the effect of Sclerostin antibodies on the healing of a tendongraft in a bone tunnel. The proposed work is innovative because it will create a tissue-engineered scaffold capable of supporting the induction of BMSCs differentiation into a ligamentous and fibrocartilaginous tissue while being able to withstand the biomechanical demands, enhancing the bone-ligament-bone interface. This will be stuies that aims to bring important knowledge to further development of needed innovative treatments to improve long term outcome for intra-articular ligament injuries.