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.

Streptococcus agalactiae (GBS) PNA FISH -tekniikan soveltuminen B-ryhmän streptokokin tunnistukseen

B-ryhmän beetahemolyyttinen streptokokki (GBS = Group B Streptococcus, Streptococcus agalactiae)aiheuttaa vakavia infektioita yleensä astasyntyneillä. Tartunta saadaan yleensä synnytyskanavasta ja riskitekijöinä ovat muun muassa keskosuus, ennenaikainen lapsivedenmeno ja äidin runsas Bstreptokokkikolonisaatio emättimessä. Bakteerin tunnistukseen käytetään tällä hetkellä viljelytekniikkaa, jonka tulos saadaan vasta 24-48 tunnin kuluttua. Opinnäytetyöni tarkoituksena on tutkia uutta ja nopeampaa tunnistusmenetelmää: GBS PNA FISH - tekniikkaa (Peptide Nucleic Acid Fluorescence in Situ Hybridization). Tarkoituksena on tutkia tekniikan spesifiteettiä ja sensitiviteettiä. Tekniikan spesifiteettiä tutkitaan B-ryhmän beetahemolyyttisellä streptokokilla sekä kuudella muulla emättimen normaaliflooraan kuuluvalla bakteerilajilla. Yhteensä bakteerikantoja on tutkimuksessa mukana 48 kappaletta. Tämän lisäksi tutkitaan myös tekniikan sensitiviteettiä, jota tutkitaan bakteereista tehdyn laimennossarjan avulla. Sensitiviteetti tutkitaan bakteeriseoksesta, jonne on B-ryhmän beetahemolyyttisen streptokokin lisäksi lisätty muita emättimen normaaliflooran bakteereita. Lisäksi sensitiviteetti tutkitaan pelkällä B-ryhmän beetahemolyyttisellä streptokokilla käyttäen sekä normaalia että bakteerin rikastusmenetelmää. Testeistä saadut tulokset tulkitaan fluoresenssimikroskoopin avulla. GBS PNA FISH -tekniikan spesifiteetti todettiin erittäin hyväksi. Tekniikka tunnisti kaikki B-ryhmän beetahemolyyttiset streptokokit positiivisiksi ja kaikki muut lajit antoivat negatiivisen tuloksen. B-streptokokin positiivisuus oli erotettavissa mikroskopoitaessa vahvana fluoresointina, kun taas muut lajit eivät fluoresoineet lainkaan. GBS PNA FISH -tekniikan sensitiivisyyden tulokset eivät kuitenkaan täyttäneet odotuksia. Ainoastaan bakteerin rikastusmenetelmällä saadut tulokset olivat loistavia, mutta bakteeriseoksella ja pelkällä B-ryhmän beetahemolyyttisellä streptokokilla saadut tulokset olivat lähes olemattomia. Rikastusmenetelmän kaikki laimennokset fluoresoivat positiivisina, kun taas muissa tapauksissa vain vahvin liuos antoi jonkinlaista positiivista fluoresointia. GBS PNA FISH -tekniikan spesifiteetti todettiin hyväksi. Tekniikan sensitiviteetti ei kuitenkaan vastaa käyttötarkoitusta ja todellisessa tilanteessa tekniikka ei pystyisi tunnistamaan sille spesifistä bakteeria muiden bakteerien joukosta.

Utilisation of reactive lysine from meat and bone meals of different ash content by growing-finishing pigs

Selostus: Tuhkapitoisuuden vaikutus lihaluujauhon reaktiivisen lysiinin hyväksikäyttöön lihasioilla

Development of Craniofacial Bone Defect Reconstruction Implant Based on Fibre-Reinforced Composite with Photopolymerisable Resin Systems. Experimental studies in vitro and in vivo.

Reconstruction of defects in the craniomaxillofacial (CMF) area has mainly been based on bone grafts or metallic fixing plates and screws. Particularly in the case of large calvarial and/or craniofacial defects caused by trauma, tumours or congenital malformations, there is a need for reliable reconstruction biomaterials, because bone grafts or metallic fixing systems do not completely fulfill the criteria for the best possible reconstruction methods in these complicated cases. In this series of studies, the usability of fibre-reinforced composite (FRC) was studied as a biostable, nonmetallic alternative material for reconstructing artificially created bone defects in frontal and calvarial areas of rabbits. The experimental part of this work describes the different stages of the product development process from the first in vitro tests with resin-impregnated fibrereinforced composites to the in vivo animal studies, in which this FRC was tested as an implant material for reconstructing different size bone defects in rabbit frontal and calvarial areas. In the first in vitro study, the FRC was polymerised in contact with bone or blood in the laboratory. The polymerised FRC samples were then incubated in water, which was analysed for residual monomer content by using high performance liquid chromatography (HPLC). It was found that this in vitro polymerisation in contact with bone and blood did not markedly increase the residual monomer leaching from the FRC. In the second in vitro study, different adhesive systems were tested in fixing the implant to bone surface. This was done to find an alternative implant fixing system to screws and pins. On the basis of this study, it was found that the surface of the calvarial bone needed both mechanical and chemical treatments before the resinimpregnated FRC could be properly fixed onto it. In three animal studies performed with rabbit frontal bone defects and critical size calvarial bone defect models, biological responses to the FRC implants were evaluated. On the basis of theseevaluations, it can be concluded that the FRC, based on E-glass (electrical glass) fibres forming a porous fibre veil enables the ingrowth of connective tissues to the inner structures of the material, as well as the bone formation and mineralization inside the fibre veil. Bone formation could be enhanced by using bioactive glass granules fixed to the FRC implants. FRC-implanted bone defects healed partly; no total healing of defects was achieved. Biological responses during the follow-up time, at a maximum of 12 weeks, to resin-impregnated composite implant seemed to depend on the polymerization time of the resin matrix of the FRC. Both of the studied resin systems used in the FRC were photopolymerised and the heat-induced postpolymerisation was used additionally.

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.

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.

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.

O Bone Jesu

Soitinnus: Sekakuoro.

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.

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.