On magnesium-containing implants for bone applications

Sammanfattning: The biomedical technologies for bone application are employed in millions of patients every year to restore function and aesthetics following trauma, diseases and congenital deformities. They achieved significant advancements in the last decades and have resulted in the development of implants that function for long periods of time. However, some fundamental clinical challenges still remain and are exacerbated by the aging of the population and by the increased life expectancy of the patients.First of all, permanent implants, despite having very high success rates, still face the risk for marginal bone loss and peri-implant osteolysis in some number of cases. Strategies to fasten, to strengthen and to maintain the bone integration of these implants are desired to enhance the implant clinical performances especially in situation of compromised bone. Secondly, the fixation of fractures and the repair of bone defects are required in a large number of clinical situations, where the intrinsic ability of bone to repair itself is limited. A constantly advocated requirement for osteosynthetic devices is the biodegradability, to avoid a second surgery for implant removal or the permanence of the device in the body for long time, with possible adverse effects. However, especially for osteosynthesis devices, materials that possess adequate mechanical properties for load-bearing applications and that biodegrade upon the substitution of new healthy osseous tissue are not yet available. Magnesium (Mg) is a material that offers potential benefits in these clinical issues. Magnesium is a natural component of the human body, which is involved in numerous enzymatic reactions and metabolic processes; thus, it is tolerated at high levels. It has a prominent role in bone homeostasis and bone health in general and it is considered bioactive, osteoconductive and angiogenetic. Therefore it could be applied as a doping agent to permanent implants and bone grafts, to increase their osseointegration. In addition, magnesium is potentially unique in the field of orthopaedic and cranio-maxillofacial surgery because it provides the mechanical properties of metals, although with an elastic modulus closer to that of cortical bone, and at the same time it degrades under physiological conditions in non-toxic by-products. Based on these clinical needs and on these observations, one aim of the current thesis was to explore the effects of the local release of Mg ions directly at the peri-implant sites on the osseointegration of titanium implants in healthy bone and in bone compromised by osteoporosis. In particular, it was of interest to attempt to elucidate the molecular and biochemical pathways that were stimulated in the peri-implant tissues by the presence of Mg ions and to correlate those to biomechanical and histomorphometrical observation. The other aim of this thesis was to characterize in vivo the degradation behaviour of 3 Mg-alloys tailored for biodegradable osteosynthesis devices and their associated bone response. In Study I to IV, the effects of the local release of Mg ions on the osseointegration of titanium implants in both healthy and osteoporotic bone were investigated. Mg ions were loaded into engineered mesoporous titanium dioxide (TiO2) carriers coated onto titanium implants. Mesoporous films acted as reservoir of drugs and bioactive substances and released them directly at the implant interface in a sustained fashion. After surface characterization of the mesoporous carriers with and without Mg ions by means of scanning electron microscopy (SEM), optical light interferometry (IFM) and atomic force microscopy (AFM), the same types of implants were implanted in animal models.In Study I, Mg-loaded implants were placed in the hind limb of rabbits for 3 weeks and examined with biomechanical analysis and histology. The results suggested that the increased local availability of Mg could accelerate and strengthen the early bone fixation of titanium implants.In Study II, the activation of biological pathways of bone healing and osseointegration of Mg-releasing implants installed in the rabbit tibia model was investigated at the gene level by means of real-time polymerase chain reaction (qPCR) after 3 weeks in vivo. The results found that several osteogenic markers (OC, RUNX-2, IGF-1) were significantly up-regulated in the presence of Mg during the first weeks of healing. This finding was correlated with the histological results, since significantly more threads for the Mg-doped implants were filled with new bone compared to the TiO2 implants without Mg. In Study III, the performance of Mg-loaded implants in bone was studied at a longer healing time of 6 weeks. It was found that the effects of Mg release are prominent in the early healing phases than compared to the later healing, presumably due to the rapid mobilization of the Mg ions from the coatings. In fact, the expression of osteogenic genes in the bone around control implants were dominantly expressed approximately 3 weeks after the dominant expression in the Mg-loaded group. Within the limitation of the observed healing period, no signs of increased inflammation and activation of bone remodelling were triggered by Mg release.In Study IV, the potential benefits of the local administration of Mg ions on implant osseointegration were tested in ovariectomized rats, which mimicked osteoporotic conditions. The presence of Mg-doped implants in osteoporotic subjects induced a significantly faster new bone formation compared to Ti controls and the activation of BMP6, an important anabolic agent that is normally suppressed in osteoporosis. In addition, other osteogenic factors, such as VEGF, were up-regulated in presence of Mg. In Study V, 3 recently developed Mg-alloys intended as temporary materials for osthesynthesis applications were tested in vivo to evaluate their degradation behaviour and the response they elicited in tissues. Mg-2Ag, Mg-10Gd and Mg-4Y-3RE in the form of mini-screws were implanted in the tibia and femur of rats for 4 and 12 weeks. Their degradation rates were investigated by means of high-resolution synchrotron-based micro computed tomography (SRµCT) and by histological sectioning. The tissue reaction to the different materials was analyzed both on histology and on 3D reconstructions of the bone-implant samples. In addition, the chemical composition of the degradation layers was assessed with Electron Probe Micro Analysis (EPMA). Finally, the expression of genes in the tissues in proximity of the mini-screws was investigated by means of qPCR employing a super-array technique.The SRµCT enabled the identification of the degradation layers, the original metal and the bone, thanks to the high spatial and density resolution. The 3-months degradation rates were similar for all materials, but the behaviour of the degradation products differed. The products of Mg-2Ag underwent rapid solubilisation. The rapid loss of sample integrity for this material led to fibrous encapsulation, rather than the desired osseous encapsulation. In the other 2 alloys, the degradation layers deposited in the same shape as the original screws and were mainly stable. That allowed the growth of bone in direct contact with the surfaces of the degradation products and they were osseointegrated at the 3-month healing time. That was confirmed on the histological slides. In addition, the chemical analysis revealed that the degradation products of the alloys were not formed by Mg, but contained Ca, P, C and O in similar amount to the surrounding bone The combination of histological, tomographic and chemical images provided new insight on the nature of the bone-to-implant interface and of the degradation products, which appeared to have great similarities to the host bone. Finally, the analysis of the genes expressed in the peri-implant bone, showed up-regulation of several genes related to osteogenesis around Mg implants compared to Ti ones. In conclusion, this thesis demonstrated that Mg is a suitable doping agent to increase the bone encapsulation of endosseous implants, especially at the early stages of healing and in particular in osteoporotic subjects. That is desirable to shorten the healing period and when early implant loading is considered an option. In addition, Mg-10Gd and Mg-4Y-3RE are biodegradable alloys with a degradation rate and behaviour that is suggested to be suitable for the new bone regeneration and the bone encapsulation.