Repair of segmental bone defects with fiber-reinforced composite: a study of material development and an animal model on rabbits

The Repair of segmental defects in load-bearing long bones is a challenging task because of the diversity of the load affecting the area; axial, bending, shearing and torsional forces all come together to test the stability/integrity of the bone. The natural biomechanical requirements for bone restorative materials include strength to withstand heavy loads, and adaptivity to conform into a biological environment without disturbing or damaging it. Fiber-reinforced composite (FRC) materials have shown promise, as metals and ceramics have been too rigid, and polymers alone are lacking in strength which is needed for restoration. The versatility of the fiber-reinforced composites also allows tailoring of the composite to meet the multitude of bone properties in the skeleton. The attachment and incorporation of a bone substitute to bone has been advanced by different surface modification methods. Most often this is achieved by the creation of surface texture, which allows bone growth, onto the substitute, creating a mechanical interlocking. Another method is to alter the chemical properties of the surface to create bonding with the bone – for example with a hydroxyapatite (HA) or a bioactive glass (BG) coating. A novel fiber-reinforced composite implant material with a porous surface was developed for bone substitution purposes in load-bearing applications. The material’s biomechanical properties were tailored with unidirectional fiber reinforcement to match the strength of cortical bone. To advance bone growth onto the material, an optimal surface porosity was created by a dissolution process, and an addition of bioactive glass to the material was explored. The effects of dissolution and orientation of the fiber reinforcement were also evaluated for bone-bonding purposes. The Biological response to the implant material was evaluated in a cell culture study to assure the safety of the materials combined. To test the material’s properties in a clinical setting, an animal model was used. A critical-size bone defect in a rabbit’s tibia was used to test the material in a load-bearing application, with short- and long-term follow-up, and a histological evaluation of the incorporation to the host bone. The biomechanical results of the study showed that the material is durable and the tailoring of the properties can be reproduced reliably. The Biological response - ex vivo - to the created surface structure favours the attachment and growth of bone cells, with the additional benefit of bioactive glass appearing on the surface. No toxic reactions to possible agents leaching from the material could be detected in the cell culture study when compared to a nontoxic control material. The mechanical interlocking was enhanced - as expected - with the porosity, whereas the reinforcing fibers protruding from the surface of the implant gave additional strength when tested in a bone-bonding model. Animal experiments verified that the material is capable of withstanding load-bearing conditions in prolonged use without breaking of the material or creating stress shielding effects to the host bone. A Histological examination verified the enhanced incorporation to host bone with an abundance of bone growth onto and over the material. This was achieved with minimal tissue reactions to a foreign body. An FRC implant with surface porosity displays potential in the field of reconstructive surgery, especially regarding large bone defects with high demands on strength and shape retention in load-bearing areas or flat bones such as facial / cranial bones. The benefits of modifying the strength of the material and adjusting the surface properties with fiber reinforcement and bone-bonding additives to meet the requirements of different bone qualities are still to be fully discovered.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.

Methods for construction and analysis of computational models in systems biology : applications to the modelling of the heat shock response and the self-assembly of intermediate filaments

Systems biology is a new, emerging and rapidly developing, multidisciplinary research field that aims to study biochemical and biological systems from a holistic perspective, with the goal of providing a comprehensive, system- level understanding of cellular behaviour. In this way, it addresses one of the greatest challenges faced by contemporary biology, which is to compre- hend the function of complex biological systems. Systems biology combines various methods that originate from scientific disciplines such as molecu- lar biology, chemistry, engineering sciences, mathematics, computer science and systems theory. Systems biology, unlike “traditional” biology, focuses on high-level concepts such as: network, component, robustness, efficiency, control, regulation, hierarchical design, synchronization, concurrency, and many others. The very terminology of systems biology is “foreign” to “tra- ditional” biology, marks its drastic shift in the research paradigm and it indicates close linkage of systems biology to computer science. One of the basic tools utilized in systems biology is the mathematical modelling of life processes tightly linked to experimental practice. The stud- ies contained in this thesis revolve around a number of challenges commonly encountered in the computational modelling in systems biology. The re- search comprises of the development and application of a broad range of methods originating in the fields of computer science and mathematics for construction and analysis of computational models in systems biology. In particular, the performed research is setup in the context of two biolog- ical phenomena chosen as modelling case studies: 1) the eukaryotic heat shock response and 2) the in vitro self-assembly of intermediate filaments, one of the main constituents of the cytoskeleton. The range of presented approaches spans from heuristic, through numerical and statistical to ana- lytical methods applied in the effort to formally describe and analyse the two biological processes. We notice however, that although applied to cer- tain case studies, the presented methods are not limited to them and can be utilized in the analysis of other biological mechanisms as well as com- plex systems in general. The full range of developed and applied modelling techniques as well as model analysis methodologies constitutes a rich mod- elling framework. Moreover, the presentation of the developed methods, their application to the two case studies and the discussions concerning their potentials and limitations point to the difficulties and challenges one encounters in computational modelling of biological systems. The problems of model identifiability, model comparison, model refinement, model inte- gration and extension, choice of the proper modelling framework and level of abstraction, or the choice of the proper scope of the model run through this thesis.

Disturbance and ecosystem functioning : the role of changes in benthic biological traits

Rapid changes in biodiversity are occurring globally, as a consequence of anthropogenic disturbance. This has raised concerns, since biodiversity is known to significantly contribute to ecosystem functions and services. Marine benthic communities participate in numerous functions provided by soft-sedimentary ecosystems. Eutrophication-induced oxygen deficiency is a growing threat against infaunal communities, both in open sea areas and in coastal zones. There is thus a need to understand how such disturbance affects benthic communities, and what is lost in terms of ecosystem functioning if benthic communities are harmed. In this thesis, the status of benthic biodiversity was assessed for the open Baltic Sea, a system severely affected by broad-scale hypoxia. Long-term monitoring data made it possible to establish quantitative biodiversity baselines against which change could be compared. The findings show that benthic biodiversity is currently severely impaired in large areas of the open Baltic Sea, from the Bornholm Basin to the Gulf of Finland. The observed reduction in biodiversity indicates that benthic communities are structurally and functionally impoverished in several of the sub-basins due to the hypoxic stress. A more detailed examination of disturbance impacts (through field studies and -experiments) on benthic communities in coastal areas showed that changes in benthic community structure and function took place well before species were lost from the system. The degradation of benthic community structure and function was directed by the type of disturbance, and its specific temporal and spatial characteristics. The observed shifts in benthic trait composition were primarily the result of reductions in species’ abundances, or of changes in demographic characteristics, such as the loss of large, adult bivalves. Reduction in community functions was expressed as declines in the benthic bioturbation potential and in secondary biomass production. The benthic communities and their degradation accounted for a substantial proportion of the changes observed in ecosystem multifunctionality. Individual ecosystem functions (i.e. measures of sediment ecosystem metabolism, elemental cycling, biomass production, organic matter transformation and physical structuring) were observed to differ in their response to increasing hypoxic disturbance. Interestingly, the results suggested that an impairment of ecosystem functioning could be detected at an earlier stage if multiple functions were considered. Importantly, the findings indicate that even small-scale hypoxic disturbance can reduce the buffering capacity of sedimentary ecosystem, and increase the susceptibility of the system towards further stress. Although the results of the individual papers are context-dependent, their combined outcome implies that healthy benthic communities are important for sustaining overall ecosystem functioning as well as ecosystem resilience in the Baltic Sea.

Physical Attractiveness as a Signal of Biological Quality

It has been commonly thought that standards of beauty are arbitrary cultural conventions that vary between cultures and time. In my thesis I found that it is not so. Instead, I show that attractiveness and preferred traits serve as cues to phenotypic qualities that provide selective benefits for those who choose their mates based on these criteria. In the first study I show that attractive men have a stronger antibody response to the hepatitis b vaccine and higher levels of testosterone than their less attractive peers. Men with low levels of testosterone also tend to have high levels of the stress hormone cortisol, suggesting that their immune responses may have been inhibited by stress hormones. Thus, facial attractiveness may serve as an honest cue of the strength of immune defence in men. In the second study, I show that the attractiveness of the male body is also a cue of better immunity. In addition, I show that adiposity, both in men’s faces and bodies, is a better cue of the strength of immunity and attractiveness than of masculinity. In the third study, I test the preferences of women from 13 countries for facial cues of testosterone and cortisol. I show that there is cross-cultural variation in women’s preference for cues of testosterone and cortisol in male faces. I found a relationship between the health of a nation and women’s preferences for cues of testosterone in the male face and the interaction between preferences for cues of testosterone and cortisol. I show also a relationship between preferences for cues of testosterone and a societal-level measure of parasite stress. Thus, it seems that societal-level ecological factors influence the relative value of traits as revealed by combinations of testosterone and stress hormones. In the fourth study, I show that women’s immune responsiveness (amount of antibodies produced) does not predict facial attractiveness. Instead, plasma cortisol level is negatively associated with attractiveness, indicating that stressed women look less attractive. Fat percentage is curvilinearly associated with facial attractiveness, indicating that being too thin or too fat reduces attractiveness. This study suggests that in contrast to men, facial attractiveness in women does not indicate the strength of immune defence, but is associated with other aspects of long-term health and fertility: circulating levels of the stress hormone cortisol and the percentage of body fat. In the last study I show that the attractiveness of men’s body odor is positively correlated with stress hormone levels, suggesting also that the attractiveness of body odors may signal the phenotypic quality of males to females. However, the attractiveness of men’s body odor was not associated with testosterone levels. My thesis suggests that the standard of beauty is not in the eye of the beholder. Instead, our standard of beauty is hardwired in our brains by genes that are selected by natural selection and also influenced by current environmental conditions.