Macroporous Scaffolds for Bone Engineering. Studies on Cell Culture and Ectopic Bone Formation

Bone engineering is a rapidly developing area of reconstructive medicine where bone inducing factors and/or cells are combined with a scaffold material to regenerate the structure and function of the original tissue. The aim of this study was to compare the suitability of different macroporous scaffold types for bone engineering applications. The two scaffold categories studied were a) the mechanically strong and stable titanium fiber meshes and b) the elastic and biodegradable porous polymers. Furthermore, bioactive modifications were applied to these basic scaffold types, and their effect on the osteogenic responses was evaluated in cell culture and ectopic bone formation studies. The osteogenic phenotype of cultured cell-scaffold constructs was heightened with a sol-gel derived titania coating, but not with a mixed titania-silica coating. The latter coating also resulted in delayed ectopic bone formation in bone marrow stromal cell seeded scaffolds. However, the better bone contact in early implantation times and more even bone tissue distribution at later times indicated enhanced osteoconductivity of both the coated scaffold types. Overall, the most promising bone engineering results were obtained with titania coated fiber meshes. Elastic and biodegradable poly(ε-caprolactone/D,L-lactide) based scaffolds were also developed in this study. The degradation rates of the scaffolds in vitro were governed by the hydrophilicity of the polymer matrix, and the porous architecture was controlled by the amount and type of porogen used. A continuous phase macroporosity was obtained using a novel CaCl2 • 6H2O porogen. Dynamic culture conditions increased cell invasion, but decreased cell numbers and osteogenicity, within the scaffolds. Osteogenic differentiation in static cultures and ectopic bone formation in cell seeded scaffolds were enhanced in composites, with 30 wt-% of bioactive glass filler.

Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.

Uusintamurtumien ehkäisy kaatumisen aiheuttaman yläraajamurtuman kokeneilla yli 50-vuotiailla henkilöillä

Tarkoitus: Tarkoituksena oli selvittää murtumien tehostetun ehkäisyohjelman vaikutuksia yläraajamurtuman saaneiden yli 50-vuotiaiden henkilöiden elämäntapoihin, murtumien riskitekijöihin, kaatumisiin, kaatumisvammoihin ja elämänlaatuun. Aineisto: 219 yli 50-vuotiasta kotona asuvaa, kaatumisen seurauksena yläraajamurtuman saanutta henkilöä satunnaistettiin koe- (n = 105) ja kontrolliryhmiin (n = 114). Menetelmä: Koeohjelma sisälsi yksilöllisen, yhdessä lääkärin, terveydenhoitajan ja tutkittavan kanssa laaditun hoito- ja kuntoutussuunnitelman sekä kutsun murtumien ehkäisyn koulutusohjelmaan. Kontrolliryhmän tutkittavat saivat kehotuksen hakeutua murtumahoitajan ohjaukseen. Seuranta-aika oli 14 kuukautta. Tulokset analysoitiin ryhmissä tapahtuneina muutoksina alkuja loppumittausten välillä sekä ryhmien välisenä erona muutoksissa. Tulokset: Keskimääräinen luun tiheys lannerangassa lisääntyi koeryhmässä (p<0,001) ja kontrolliryhmässä (p = 0,038), ryhmien välinen ero ei ollut tilastollisesti merkitsevä (p = 0,134). Reisiluun kaulassa luun tiheys ei muuttunut kummassakaan ryhmässä. Päivittäinen kalsiumin saanti lisääntyi koeryhmässä keskimäärin 167 mg (p<0,001) ja kontrolliryhmässä 30 mg (p = 0,475), ryhmien välinen ero oli tilastollisesti merkitsevä (p = 0,031). Kala-ateriat ja päivittäinen D-vitamiinilisä sekä kenkien liukuesteiden säännöllinen käyttö liukkaalla lisääntyivät molemmissa ryhmissä, mutta ryhmien välillä ei ollut eroa. Viiteen tuolilta nousukertaan käytetty aika vähentyi koeryhmässä keskimäärin 0,49 sekuntia (p = 0,185) ja lisääntyi kontrolliryhmässä 0,39 (p = 0,475), ero ryhmien välillä oli tilastollisesti merkitsevä (p = 0,02). Kymmenen metrin kävelyssä ei tapahtunut tulosten keskiarvossa muutoksia kummassakaan ryhmässä. Ortostatismi vähentyi kontrolliryhmässä (p = 0,049), ja masentuneisuus vähentyi koeryhmässä (p = 0,041), mutta ryhmien välillä ei näissä ollut merkitsevää eroa. Osteoporoosilääkkeiden käyttö lisääntyi molemmissa ryhmissä, ryhmien välillä ei ollut eroa. Alkoholin käytössä oli vähäisiä mutta ei merkittäviä muutoksia. Muissa testatuissa muuttujissa, kuten liikunnan viikoittainen määrä, tupakointi, näkökyky, tasapaino ja elämänlaatu, ei ollut merkitseviä muutoksia ryhmissä tai ryhmien välillä. Kaatumisten (koeryhmä 33, kontrolliryhmä 35) ja murtumien (koeryhmä 5 ja kontrolliryhmä 3) määrissä ei myöskään ollut merkitseviä eroja ryhmien välillä. Johtopäätökset: Noin neljällä viidestä kaatumisen seurauksena yläraajamurtuman saaneista, yli 50 vuotta täyttäneistä henkilöistä oli alentunut luun tiheys, ja noin joka toisella oli lisääntynyt kaatumisriski. Kaatumisen riskitekijöiden kliiniset mittaukset olivat nopeita toteuttaa ja auttoivat ehkäisytoimenpiteiden suunnittelussa. Murtuman saaneet olivat motivoituneita murtumien riskitekijöiden selvittämiseen ja myös toteuttamaan annettua lääkehoitoa. Elämäntavoissa helpoimmin toteutuivat muutokset ruokailutottumuksissa. Liikuntatottumuksien muuttaminen sen sijaan onnistui melko huonosti. Ryhmien välillä oli merkitsevä ero ainoastaan kalsiumin päivittäisessä käytössä ja tuolilta nousutestissä. Murtumariskien tunnistaminen, niiden syiden selvittäminen ja toimenpiteiden käynnistäminen riskien vähentämiseksi tulisi kuulua jokaisen pienienergisen murtuman saaneen potilaan kokonaisvaltaiseen hyvään hoitoon.

Analytic evaluation of three-phase short circuit demagnetization and hysteresis loss risk in rotor-surface-magnet Permanent-Magnet Synchronous Machines

Permanent magnet synchronous machines (PMSM) have become widely used in applications because of high efficiency compared to synchronous machines with exciting winding or to induction motors. This feature of PMSM is achieved through the using the permanent magnets (PM) as the main excitation source. The magnetic properties of the PM have significant influence on all the PMSM characteristics. Recent observations of the PM material properties when used in rotating machines revealed that in all PMSMs the magnets do not necessarily operate in the second quadrant of the demagnetization curve which makes the magnets prone to hysteresis losses. Moreover, still no good analytical approach has not been derived for the magnetic flux density distribution along the PM during the different short circuits faults. The main task of this thesis is to derive simple analytical tool which can predict magnetic flux density distribution along the rotor-surface mounted PM in two cases: during normal operating mode and in the worst moment of time from the PM’s point of view of the three phase symmetrical short circuit. The surface mounted PMSMs were selected because of their prevalence and relatively simple construction. The proposed model is based on the combination of two theories: the theory of the magnetic circuit and space vector theory. The comparison of the results in case of the normal operating mode obtained from finite element software with the results calculated with the proposed model shows good accuracy of model in the parts of the PM which are most of all prone to hysteresis losses. The comparison of the results for three phase symmetrical short circuit revealed significant inaccuracy of the proposed model compared with results from finite element software. The analysis of the inaccuracy reasons was provided. The impact on the model of the Carter factor theory and assumption that air have permeability of the PM were analyzed. The propositions for the further model development are presented.

Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

Bone Mineral Accrual in Physically Active Girls. With Special Reference to Reduction in Physical Activity Level and Use of Oral Contraceptives

Adolescence is an important time for acquiring high peak bone mass. Physical activity is known to be beneficial to bone development. The effect of estrogen-progestin contraceptives (EPC) is still controversial. Altogether 142 (52 gymnasts, 46 runners, and 42 controls) adolescent women participated in this study, which is based on two 7-year (n =142), one 6-year (n =140) and one 4-year (n =122) follow-ups. Information on physical activity, menstrual history, sexual maturation, nutrition, living habits and health status was obtained through questionnaires and interviews. The bone mineral density (BMD) and content (BMC) of lumbar spine (LS) and femoral neck (FN) were measured by dual- energy X-ray absoptiometry. Calcaneal sonographic measurements were also made. The physical activity of the athletes participating in this study decreased after 3-year follow-up. High-impact exercise was beneficial to bones. LS and FN BMC was higher in gymnasts than in controls during the follow-up. Reduction in physical activity had negative effects on bone mass. LS and FN BMC increased less in the group having reduced their physical activity more than 50%, compared with those continuing at the previous level (1.69 g, p=0.021; 0.14 g, p=0.015, respectively). The amount of physical activity was the only significant parameter accounting for the calcaneal sonography measurements at 6-year follow-up (11.3%) and reduced activity level was associated with lower sonographic values. Long-term low-dose EPC use seemed to prevent normal bone mass acquisition. There was a significant trend towards a smaller increase in LS and FN BMC among long-term EPC users. In conclusion, this study confirms that high-impact exercise is beneficial to bones and that the benefits are partly maintained even after a clear reduction in training level at least for 4 years. Continued exercise is needed to retain all acquired benefits. The bone mass gained and maintained can possibly be maximized in adolescence by implementing high-impact exercise for youngsters. The peak bone mass of the young women participating in the study may be reached before the age of 20. Use of low-dose EPCs seems to suppress normal bone mass acquisition.

Wood as a model material for medical biomaterials. In vivo and in vitro studies with bone and Betula pubescens Ehrh

Novel biomaterials are needed to fill the demand of tailored bone substitutes required by an ever‐expanding array of surgical procedures and techniques. Wood, a natural fiber composite, modified with heat treatment to alter its composition, may provide a novel approach to the further development of hierarchically structured biomaterials. The suitability of wood as a model biomaterial as well as the effects of heat treatment on the osteoconductivity of wood was studied by placing untreated and heat‐treated (at 220 C , 200 degrees and 140 degrees for 2 h) birch implants (size 4 x 7mm) into drill cavities in the distal femur of rabbits. The follow‐up period was 4, 8 and 20 weeks in all in vivo experiments. The flexural properties of wood as well as dimensional changes and hydroxyl apatite formation on the surface of wood (untreated, 140 degrees C and 200 degrees C heat‐treated wood) were tested using 3‐point bending and compression tests and immersion in simulated body fluid. The effect of premeasurement grinding and the effect of heat treatment on the surface roughness and contour of wood were tested with contact stylus and non‐contact profilometry. The effects of heat treatment of wood on its interactions with biological fluids was assessed using two different test media and real human blood in liquid penetration tests. The results of the in vivo experiments showed implanted wood to be well tolerated, with no implants rejected due to foreign body reactions. Heat treatment had significant effects on the biocompatibility of wood, allowing host bone to grow into tight contact with the implant, with occasional bone ingrowth into the channels of the wood implant. The results of the liquid immersion experiments showed hydroxyl apatite formation only in the most extensively heat‐treated wood specimens, which supported the results of the in vivo experiments. Parallel conclusions could be drawn based on the results of the liquid penetration test where human blood had the most favorable interaction with the most extensively heat‐treated wood of the compared materials (untreated, 140 degrees C and 200 degrees C heat‐treated wood). The increasing biocompatibility was inferred to result mainly from changes in the chemical composition of wood induced by the heat treatment, namely the altered arrangement and concentrations of functional chemical groups. However, the influence of microscopic changes in the cell walls, surface roughness and contour cannot be totally excluded. The heat treatment was hypothesized to produce a functional change in the liquid distribution within wood, which could have biological relevance. It was concluded that the highly evolved hierarchical anatomy of wood could yield information for the future development of bulk bone substitutes according to the ideology of bioinspiration. Furthermore, the results of the biomechanical tests established that heat treatment alters various biologically relevant mechanical properties of wood, thus expanding the possibilities of wood as a model material, which could include e.g. scaffold applications, bulk bone applications and serving as a tool for both mechanical testing and for further development of synthetic fiber reinforced composites.

Bone Health, Osteoporosis and Fracture Risk in Neurofibromatosis 1 - An Emphasis on Osteoclasts

Neurofibromatosis 1 (NF1) is an autosomal dominant hereditary syndrome, affecting skin, neural tissues and skeleton. Hallmarks of NF1 include benign cutaneous neurofibroma tumors, pigmentation lesions on the skin and in the iris, learning disabilities and predisposition to selected malignancies. Low bone mineral density (BMD) and osteopenia/osteoporosis are common in NF1. Osteoporosis is a systemic disorder characterized by low bone mineral density and increased fracture risk. Treatment of osteoporosis aims to prevent falls and decrease fracture risk. Osteoporosis is diagnosed in adults by measuring BMD and evaluating clinical risk factors of the patient. Bone turnover is a process of old bone resorbed by osteoclasts and new bone formed by osteoblasts. Multinuclear osteoclasts are derived from osteoclast progenitors, which can be isolated from peripheral blood. Osteoclast progenitors were isolated from 17 NF1 patients and healthy controls, and cultured in vitro to osteoclasts. NF1 osteoclasts are hyperactive, displaying increased differentiation and resorption capacity, abnormal morphology and tolerance to serum deprivation compared to control osteoclasts. These findings expanded the study to evaluate the effects of bisphosphonates, drugs designed to treat osteoporosis, in osteoclasts derived from blood samples of 20 NF1 and control persons. The number of control osteoclasts was expectedly reduced after bisphosphonate treatment. However, NF1 osteoclasts tolerated the apoptotic effect of alendronate, zoledronic acid and clodronate in vitro compared to controls. NF1-related osteoporosis was found in ~20 % of the patients, and selected laboratory parameters were measured. Patients with NF1 have increased levels of serum CTX and PINP, reflecting increased bone turnover in vivo. BMD decreases progressively in NF1 as evaluated in 19 NF1 patients 12 years after their initial BMD measurement. Patients with NF1-related osteopenia often progress to osteoporosis. This was found in patients aged 37-76.

Flexible multibody approach in bone strain estimation during physical activity:

Bone strain plays a major role as the activation signal for the bone (re)modeling process, which is vital for keeping bones healthy. Maintaining high bone mineral density reduces the chances of fracture in the event of an accident. Numerous studies have shown that bones can be strengthened with physical exercise. Several hypotheses have asserted that a stronger osteogenic (bone producing) effect results from dynamic exercise than from static exercise. These previous studies are based on short-term empirical research, which provide the motivation for justifying the experimental results with a solid mathematical background. The computer simulation techniques utilized in this work allow for non-invasive bone strain estimation during physical activity at any bone site within the human skeleton. All models presented in the study are threedimensional and actuated by muscle models to replicate the real conditions accurately. The objective of this work is to determine and present loading-induced bone strain values resulting from physical activity. It includes a comparison of strain resulting from four different gym exercises (knee flexion, knee extension, leg press, and squat) and walking, with the results reported for walking and jogging obtained from in-vivo measurements described in the literature. The objective is realized primarily by carrying out flexible multibody dynamics computer simulations. The dissertation combines the knowledge of finite element analysis and multibody simulations with experimental data and information available from medical field literature. Measured subject-specific motion data was coupled with forward dynamics simulation to provide natural skeletal movement. Bone geometries were defined using a reverse engineering approach based on medical imaging techniques. Both computed tomography and magnetic resonance imaging were utilized to explore modeling differences. The predicted tibia bone strains during walking show good agreement with invivo studies found in the literature. Strain measurements were not available for gym exercises; therefore, the strain results could not be validated. However, the values seem reasonable when compared to available walking and running invivo strain measurements. The results can be used for exercise equipment design aimed at strengthening the bones as well as the muscles during workout. Clinical applications in post fracture recovery exercising programs could also be the target. In addition, the methodology introduced in this study, can be applied to investigate the effect of weightlessness on astronauts, who often suffer bone loss after long time spent in the outer space.

Inverting ionospheric D-region electron density pro-file from riometer measurement

The aim of this work is to invert the ionospheric electron density profile from Riometer (Relative Ionospheric opacity meter) measurement. The newly Riometer instrument KAIRA (Kilpisjärvi Atmospheric Imaging Receiver Array) is used to measure the cosmic HF radio noise absorption that taking place in the D-region ionosphere between 50 to 90 km. In order to invert the electron density profile synthetic data is used to feed the unknown parameter Neq using spline height method, which works by taking electron density profile at different altitude. Moreover, smoothing prior method also used to sample from the posterior distribution by truncating the prior covariance matrix. The smoothing profile approach makes the problem easier to find the posterior using MCMC (Markov Chain Monte Carlo) method.