Multi-analyte detection for biological fluids. Towards continous monitoring of glucose, ionized calcium and pH using a viscometric affinity biosensor.

We present a viscometric affinity biosensor that can potentially allow continuous multi-analyte monitoring in biological fluids like blood or plasma. The sensing principle is based on the detection of viscosity changes of a polymeric solution which has a selective affinity for the analyte of interest. The chemico-mechanical sensor incorporates an actuating piezoelectric diaphragm, a sensing piezoelectric diaphragm and a flow-resisting microchannel for viscosity detection. A free-standing Anodic Alumina Oxide (AAO) porous nano-membrane is used as selective interface. A glucose-sensitive sensor was fabricated and extensively assessed in buffer solution. The sensor reversibility, stability and sensitivity were excellent during at least 65 hours. Results showed also a good degree of stability for a long term measurement (25 days). The sensor behaviour was furthermore tested in fetal bovine serum (FBS). The obtained results for glucose sensing are very promising, indicating that the developed sensor is a candidate for continuous monitoring in biological fluids. Sensitive solutions for ionized calcium and pH are currently under development and should allow multi-analyte sensing in the near future.

Optimal Mechanical PulpingProcesses for Eucalyptus, Acacia and Birch

Tässä diplomityössä tutkittiin ja vertailtiin eukalyptuksen, akaasian ja koivun kemimekaanista kuiduttamista ja valkaisua. Yleensä näitä puulajeja käytetään sellun keittoon. Puulajit eroavat toisistaan kasvupaikan ja kuiturakenteen osalta. Eukalyptus ja akaasia ovat niin sanottuja trooppisia lehtipuita, kun taas koivu kasvaa pohjoisilla vyöhykkeillä. Koivulla on kookkaimmat kuidut ja akaasialla pienimmät kuidut. Myös näiden lajien putkilot eroavat toisistaan. Koivun putkilot ovat pitkiä ja kapeita, kun taas eukalyptuksen ja akaasian putkilot ovat lyhyitä ja leveitä. Prosessiksi valittiin kaksivaiheinen APMP-prosessi. Koeajot tehtiinKeskuslaboratorio Oy:ssä. Massoille asetettiin seuraavat tavoitteet: freeness 150-200 ml ja vaaleus 80 %ISO. Eukalyptukselle ja koivulle tehtiin kaksi erilaista impregnointisarjaa, mutta akaasialle vain yksi. Jauhatuksen viimeisessä vaiheessa kokeiltiin myös jauhinvalkaisua. Jauhatuksen energiankulutus oli korkea varsinkin eukalyptuksella ja akaasialla. Jotta energiankulutus saataisiin pienemmäksi, tulisi käyttää enemmän lipeää, mutta se johtaa alkalitummumiseen. Lopuksi massat valkaistiin laboratoriossa. Eukalyptus ja koivu pystyttiin valkaisemaan vaaleuteen 80 %ISO, mutta eukalyptuksen valkaisu vaati enemmän peroksidia kuin koivun valkaisu. Akaasian lähtövaaleus oli niin alhainen, ettei siinä päästy tavoitevaaleuteen. Eukalyptuksella on parempi valonsironta ja paremmat lujuusominaisuudet kuin koivulla. Kemimekaanista massaa voidaan käyttää hienopaperissa parantamassa jäykkyyttä, bulkkia ja valonsirontaa, mutta usein ongelmana on alhainen vaaleus ja huono vaaleuden pysyvyys. Kemimekaanista massaa voidaankäyttää missä tahansa mekaanisissa painopapereissa. Mekaanisissa painopapereissa kemimekaanisella lehtipuumassalla voidaan korvata mekaanista havupuumassaa. Akaasia on niin tummaa, ettei sitä voida käyttää korkeavaaleuksisiin papereihin. Eukalyptus ja koivu ovat vaaleampia ja helpompia valkaista kuin akaasia, mutta myös niillä on niin huono vaaleudenpysyvyys että käyttö hienopapereissa on rajoittunutta. Mekaanisille eukalyptus ja koivumassoille hienopaperia parempi käyttökohde on mekaaniset painopaperit, kuten MWC-paperi.

Flexible Multibody Simulation Approach in the Dynamic Analysis of Bone Strains Activity

The objective of this study is to show that bone strains due to dynamic mechanical loading during physical activity can be analysed using the flexible multibody simulation approach. Strains within the bone tissue play a major role in bone (re)modeling. Based on previous studies, it has been shown that dynamic loading seems to be more important for bone (re)modeling than static loading. The finite element method has been used previously to assess bone strains. However, the finite element method may be limited to static analysis of bone strains due to the expensive computation required for dynamic analysis, especially for a biomechanical system consisting of several bodies. Further, in vivo implementation of strain gauges on the surfaces of bone has been used previously in order to quantify the mechanical loading environment of the skeleton. However, in vivo strain measurement requires invasive methodology, which is challenging and limited to certain regions of superficial bones only, such as the anterior surface of the tibia. In this study, an alternative numerical approach to analyzing in vivo strains, based on the flexible multibody simulation approach, is proposed. In order to investigate the reliability of the proposed approach, three 3-dimensional musculoskeletal models where the right tibia is assumed to be flexible, are used as demonstration examples. The models are employed in a forward dynamics simulation in order to predict the tibial strains during walking on a level exercise. The flexible tibial model is developed using the actual geometry of the subject’s tibia, which is obtained from 3 dimensional reconstruction of Magnetic Resonance Images. Inverse dynamics simulation based on motion capture data obtained from walking at a constant velocity is used to calculate the desired contraction trajectory for each muscle. In the forward dynamics simulation, a proportional derivative servo controller is used to calculate each muscle force required to reproduce the motion, based on the desired muscle contraction trajectory obtained from the inverse dynamics simulation. Experimental measurements are used to verify the models and check the accuracy of the models in replicating the realistic mechanical loading environment measured from the walking test. The predicted strain results by the models show consistency with literature-based in vivo strain measurements. In conclusion, the non-invasive flexible multibody simulation approach may be used as a surrogate for experimental bone strain measurement, and thus be of use in detailed strain estimation of bones in different applications. Consequently, the information obtained from the present approach might be useful in clinical applications, including optimizing implant design and devising exercises to prevent bone fragility, accelerate fracture healing and reduce osteoporotic bone loss.

Non-resorbable glass fibre-reinforced composite with porous surface as bone substitute material: Experimental studies in vitro and in vivo focused on bone-implant interface

The development of load-bearing osseous implant with desired mechanical and surface properties in order to promote incorporation with bone and to eliminate risk of bone resorption and implant failure is a very challenging task. Bone formation and resoption processes depend on the mechanical environment. Certain stress/strain conditions are required to promote new bone growth and to prevent bone mass loss. Conventional metallic implants with high stiffness carry most of the load and the surrounding bone becomes virtually unloaded and inactive. Fibre-reinforced composites offer an interesting alternative to metallic implants, because their mechanical properties can be tailored to be equal to those of bone, by the careful selection of matrix polymer, type of fibres, fibre volume fraction, orientation and length. Successful load transfer at bone-implant interface requires proper fixation between the bone and implant. One promising method to promote fixation is to prepare implants with porous surface. Bone ingrowth into porous surface structure stabilises the system and improves clinical success of the implant. The experimental part of this work was focused on polymethyl methacrylate (PMMA) -based composites with dense load-bearing core and porous surface. Three-dimensionally randomly orientated chopped glass fibres were used to reinforce the composite. A method to fabricate those composites was developed by a solvent treatment technique and some characterisations concerning the functionality of the surface structure were made in vitro and in vivo. Scanning electron microscope observations revealed that the pore size and interconnective porous architecture of the surface layer of the fibre-reinforced composite (FRC) could be optimal for bone ingrowth. Microhardness measurements showed that the solvent treatment did not have an effect on the mechanical properties of the load-bearing core. A push-out test, using dental stone as a bone model material, revealed that short glass fibre-reinforced porous surface layer is strong enough to carry load. Unreacted monomers can cause the chemical necrosis of the tissue, but the levels of leachable resisidual monomers were considerably lower than those found in chemically cured fibre-reinforced dentures and in modified acrylic bone cements. Animal experiments proved that surface porous FRC implant can enhance fixation between bone and FRC. New bone ingrowth into the pores was detected and strong interlocking between bone and the implant was achieved.

Application of cholesterol determination method to indirectly detect meat and bone meals in ruminant feeds

The aim of this study was to verify the presence of meat and bone meal (MBM) in ruminant feed, by identifying the cholesterol using gas chromatography with a flame ionization detector. The proposed method demonstrated precision, trueness, and capability to detect MBM in the ruminant feed.

Utilization of Meat and bone meal for energy production

At the end of the 1990s the stock breeding in the Europe was suffering from the animal disease epidemics such as Bovine spongiform encephalopathy (BSE) and foot –and mouth disease. The European Union (EU) tackled to this problem by tightening the legislation of animal by-products. At this point, rendering and fat producing industries faces new challenges, which they have to cope with in a way of trying to find alternatives to their products (animal fats and meat and bone meal). One of the most promising alternatives to utilize these products was to use them in energy production purposes. The purpose of the Thesis was to examine the utilization possibilities of Meat and bone meal (MBM) for energy production. The first part of the Thesis consists of theory part. The theory part includes evaluation of basic properties of MBM as a fertilizer and as a fuel, legislative evaluation and evaluation of different burning techniques. The second part of the Thesis consists of burning tests in Energy laboratory of LUT with different mixtures of peat and MBM. The purpose of the burning tests was to identify co-firing possibilities of peat and MBM and emission- and ash properties for peat and MBM.

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.

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Curving simulation and stability of a creep-controlled wheelset for high speed rail-vehicles

Higher travel speeds of rail vehicles will be possible by developing sophisticated top performance bogies having creep-controlled wheelsets. In this case the torque transmission between the right and the left wheel is realized by an actively controlled creep coupling. To investigate hunting stability and curving capability the linear equations of motion are written in state space notation. Simulation results are obtained with realistic system parameters from industry and various controller gains. The advantage of the „creep-controlled wheelset" is discussed by comparison the simulation results with the dynamic behaviour of the special cases „solid-axle wheelset" and „loose wheelset" (independent rotation of the wheels). The stability is also investigated with a root-locus analysis.

Experimental Studies on Non-Metallic Composite Bone Implants With a Special Reference to Staphylococcal Biofilm Infection

Non-metallic implants made of bioresorbable or biostable synthetic polymers are attractive options in many surgical procedures, ranging from bioresorbable suture anchors of arthroscopic surgery to reconstructive skull implants made of biostable fiber-reinforced composites. Among other benefits, non-metallic implants produce less interference in imaging. Bioresorbable polymer implants may be true multifunctional, serving as osteoconductive scaffolds and as matrices for simultaneous delivery of bone enhancement agents. As a major advantage for loading conditions, mechanical properties of biostable fiber-reinforced composites can be matched with those of the bone. Unsolved problems of these biomaterials are related to the risk of staphylococcal biofilm infections and to the low osteoconductivity of contemporary bioresorbable composite implants. This thesis was focused on the research and development of a multifunctional implant model with enhanced osteoconductivity and low susceptibility to infection. In addition, the experimental models for assessment, diagnostics and prophylaxis of biomaterial-related infections were established. The first experiment (Study I) established an in vitro method for simultaneous evaluation of calcium phosphate and biofilm formation on bisphenol-Aglycidyldimethacrylate and triethylenglycoldimethacrylate (BisGMA-TEGDMA) thermosets with different content of bioactive glass 45S5. The second experiment (Study II) showed no significant difference in osteointegration of nanostructured and microsized polylactide-co-glycolide/β-tricalcium phosphate (PLGA /β-TCP) composites in a minipig model. The third experiment (Study III) demonstrated that positron emission tomography (PET) imaging with the novel 68Ga labelled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) CD33 related sialic-acid immunoglobulin like lectins (Siglec-9) tracer was able to detect inflammatory response to S. epidermidis and S. aureus peri-implant infections in an intraosseous polytetrafluoroethylene catheter model. In the fourth experiment (Study IV), BisGMATEGDMA thermosets coated with lactose-modified chitosan (Chitlac) and silver nanoparticles exhibited antibacterial activity against S. aureus and P. aeruginosa strains in an in vitro biofilm model and showed in vivo biocompatibility in a minipig model. In the last experiment (Study V), a selective androgen modulator (SARM) released from a poly(lactide)-co-ε-caprolactone (PLCL) polymer matrix failed to produce a dose-dependent enhancement of peri-implant osteogenesis in a bone marrow ablation model.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Experimental model of heterotopic ossification in Wistar rats

Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues adjacent to large joints, resulting in joint mobility deficit. In order to determine which treatment techniques are more appropriate for such condition, experimental models of induced heterotopic bone formation have been proposed using heterologous demineralized bone matrix implants and bone morphogenetic protein and other tissues. The objective of the present experimental study was to identify a reliable protocol to induce HO in Wistar rats, based on autologous bone marrow (BM) implantation, comparing 3 different BM volumes and based on literature evidence of this HO induction model in larger laboratory animals. Twelve male Wistar albino rats weighing 350/390 g were used. The animals were anesthetized for blood sampling before HO induction in order to quantify serum alkaline phosphatase (ALP). HO was induced by BM implantation in both quadriceps muscles of these animals, experimental group (EG). Thirty-five days after the induction, another blood sample was collected for ALP determination. The results showed a weight gain in the EG and no significant difference in ALP levels when comparing the periods before and after induction. Qualitative histological analysis confirmed the occurrence of heterotopic ossification in all 12 EG rats. In conclusion, the HO induction model was effective when 0.35 mL autologous BM was applied to the quadriceps of Wistar rats.

Influence of transcutaneous electrical stimulation on heterotopic ossification: an experimental study in Wistar rats

Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues, resulting in joint mobility deficit and pain. Different treatment modalities have been tried to prevent HO development, but there is no consensus on a therapeutic approach. Since electrical stimulation is a widely used resource in physiotherapy practice to stimulate joint mobility, with analgesic and anti-inflammatory effects, its usefulness for HO treatment was investigated. We aimed to identify the influence of electrical stimulation on induced HO in Wistar rats. Thirty-six male rats (350-390 g) were used, and all animals were anesthetized for blood sampling before HO induction, to quantify the serum alkaline phosphatase. HO induction was performed by bone marrow implantation in both quadriceps of the animals, which were then divided into 3 groups: control (CG), transcutaneous electrical nerve stimulation (TENS) group (TG), and functional electrical stimulation (FES) group (FG) with 12 rats each. All animals were anesthetized and electrically stimulated twice per week, for 35 days from induction day. After this period, another blood sample was collected and quadriceps muscles were bilaterally removed for histological and calcium analysis and the rats were killed. Calcium levels in muscles showed significantly lower results when comparing TG and FG (P<0.001) and between TG and CG (P<0.001). Qualitative histological analyses confirmed 100% HO in FG and CG, while in TG the HO was detected in 54.5% of the animals. The effects of the muscle contractions caused by FES increased HO, while anti-inflammatory effects of TENS reduced HO.

High-impact exercise in rats prior to and during suspension can prevent bone loss

High-impact exercise has been considered an important method for treating bone loss in osteopenic experimental models. In this study, we investigated the effects of osteopenia caused by inactivity in femora and tibiae of rats subjected to jump training using the rat tail suspension model. Eight-week-old female Wistar rats were divided into five groups (n=10 each group): jump training for 2 weeks before suspension and training during 3 weeks of suspension; jump training for 2 weeks before suspension; jump training only during suspension; suspension without any training; and a control group. The exercise protocol consisted of 20 jumps/day, 5 days/week, with a jump height of 40 cm. The bone mineral density of the femora and tibiae was measured by double energy X-ray absorptiometry and the same bones were evaluated by mechanical tests. Bone microarchitecture was evaluated by scanning electron microscopy. One-way ANOVA was used to compare groups. Significance was determined as P<0.05. Regarding bone mineral density, mechanical properties and bone microarchitecture, the beneficial effects were greater in the bones of animals subjected to pre-suspension training and subsequently to training during suspension, compared with the bones of animals subjected to pre-suspension training or to training during suspension. Our results indicate that a period of high impact exercise prior to tail suspension in rats can prevent the installation of osteopenia if there is also training during the tail suspension.