Characterisation and in vitro and in vivo evaluation of supercritical-CO2-foamed ß-TCP/PLCL composites for bone applications.

Most synthetic bone grafts are either hard and brittle ceramics or paste-like materials that differ in applicability from the gold standard autologous bone graft, which restricts their widespread use. Therefore, the aim of the study was to develop an elastic, highly porous and biodegradable ß-tricalciumphosphate/poly(L-lactide-co-ε-caprolactone) (ß-TCP/PLCL) composite for bone applications using supercritical CO2 foaming. Ability to support osteogenic differentiation was tested in human adipose stem cell (hASC) culture for 21 d. Biocompatibility was evaluated for 24 weeks in a rabbit femur-defect model. Foamed composites had a high ceramic content (50 wt%) and porosity (65-67 %). After 50 % compression, in an aqueous environment at 37 °C, tested samples returned to 95 % of their original height. Hydrolytic degradation of ß-TCP/PLCL composite, during the 24-week follow-up, was very similar to that of porous PLCL scaffold both in vitro and in vivo. Osteogenic differentiation of hASCs was demonstrated by alkaline phosphatase activity analysis, alizarin red staining, soluble collagen analysis, immunocytochemical staining and qRT-PCR. In vitro, hASCs formed a pronounced mineralised collagen matrix. A rabbit femur defect model confirmed biocompatibility of the composite. According to histological Masson-Goldner's trichrome staining and micro-computed tomography, ß-TCP/PLCL composite did not elicit infection, formation of fibrous capsule or cysts. Finally, native bone tissue at 4 weeks was already able to grow on and in the ß-TCP/PLCL composite. The elastic and highly porous ß-TCP/PLCL composite is a promising bone substitute because it is osteoconductive and easy-to-use and mould intraoperatively.

Chemical interactions in composites of gellan gum and bioactive glass: self-crosslinking and in vitro dissolution.

We investigated the interactions between the organic-inorganic phases in composites and the impact on in vitro dissolution. The composite consists of a hydrogel-forming polysaccharide gellan gum (GG, organic phase) and a borosilicate bioactive glass (BAG, inorganic phase). The BAG loading in the gellan gum matrix varied from 10 to 50 wt%. While mixing GG and BAG, the ions released from BAG microparticles crosslinked with the carboxylate anions of GG. The nature of the crosslinking was assessed, and its impact on mechanical properties, swelling ratio, and enzymatic degradation profile upon immersion for up to 2 weeks was studied. Loading up to 30 wt% of BAG in GG caused an increase in mechanical properties associated with an increasing crosslinking density. At higher BAG loading, excess divalent ions and percolation of particles led to a decrease in the fracture strength and compressive modulus. Upon immersion, a decrease in the composite mechanical properties was attributed to the dissolution of the BAG and the loosening of the glass/matrix interface. The enzymatic degradation of the composites was inhibited at higher BAG loadings (40 and 50 wt%) even when the specimen was immersed for 48 h in PBS buffer with lysozyme. During in vitro dissolution in both SBF and PBS, the ions released from the glass led to the precipitation of hydroxyapatite already at day 7. In conclusion, we thoroughly discussed the in vitro stability of the GG/BAG composite and established the maximum BAG loading to enhance the GG crosslinking and mechanical properties. Based on this study, 30, 40, and 50 wt% of BAG in GG will be further investigated in an in vitro cell culture study.

Programmed water-induced shape-memory of bioabsorbable poly(D,L-lactide): activation and properties in physiological temperature.

This study reports of the novel water-induced shape-memory of bioabsorbable poly(D,L-lactide). We have developed an orientation-based programming process that generates an ability for poly(D,L-lactide) to transform its shape at 37°C in an aqueous environment without external energy and to adapt to a predefined stress level by stress generation or relaxation. In this orientation-programming process, polymer material is deformed and oriented at an elevated temperature and subsequently cooled down while retaining its deformed shape, tension, and polymer chain entanglements. At body temperature and in an aqueous environment, the shape-memory is activated by the plasticizing effect of water molecules diffused into the polymer matrix causing an entropy-driven directed relaxation of oriented and preloaded polymer chains. This plasticizing effect is clearly seen as a decrease of the onset glass transition temperature by 10-13°C. We found that γ-irradiation used for sterilizing the orientation-programmed materials strongly affected the shape-recovery rate, but not the recovery ratio. Both non-γ-irradiated and γ-irradiated sample materials showed excellent shape-recovery ratios during a ten-week test period: 94 and 97%, respectively. The orientation-programmed materials generated a predefined load in a 37°C aqueous environment when their shape-recovery was restricted, but when external tension was applied to them, they adapted to the predefined level by stress relaxation. Our results show that functionality in terms of shape-memory can be generated in bioabsorbable polymers without tailoring the polymer chain structure thus shortening the time from development of technology to its utilization in medical devices.

Cartilage Repair Capacity within a Single Full-Thickness Chondral Defect in a Porcine Autologous Matrix-Induced Chondrogenesis Model Is Affected by the Location within the Defect.

OBJECTIVE: Large articular cartilage defects are a challenge to regenerative surgery. Biomaterial scaffolds might provide valuable support for restoration of articulating surface. The performance of a composite biomaterial scaffold was evaluated in a large porcine cartilage defect. DESIGN: Cartilage repair capacity of a biomaterial combining recombinant human type III collagen (rhCo) and poly-(l/d)-lactide (PLA) was tested in a porcine model. A full-thickness chondral defect covering the majority of the weightbearing area was inflicted to the medial femoral condyle of the right knee. Spontaneous cartilage repair and nonoperated healthy animals served as controls. The animals were sacrificed after a 4-month follow-up. The repair tissue was evaluated with the International Cartilage Repair Society (ICRS) macroscopic score, ICRS II histological score, and with micro-computed tomography. Additionally, histopathological evaluation of lymph nodes and synovial samples were done for toxicological analyses. RESULTS: The lateral half of the cartilage defect in the operated groups showed better filling than the medial half. The mean overall macroscopic score for the rhCo-PLA, spontaneous, and nonoperated groups were 5.96 ± 0.33, 4.63 ± 0.42, and 10.98 ± 0.35, respectively. The overall histological appearance of the specimens was predominantly hyaline cartilage in 3 of 9 samples of the rhCo-PLA group, 2 of 8 of the spontaneous group, and 9 of 9 of the nonoperated group. CONCLUSIONS: The use of rhCo-PLA scaffold did not differ from spontaneous healing. The repair was affected by the spatial properties within the defect, as the lateral part of the defect showed better repair than the medial part, probably due to different weightbearing conditions.

Targeted exercises against hip fragility.

SUMMARY: Compared to high-impact exercises, moderate-magnitude impacts from odd-loading directions have similar ability to thicken vulnerable cortical regions of the femoral neck. Since odd-impact exercises are mechanically less demanding to the body, this type of exercise can provide a reasonable basis for devising feasible, targeted bone training against hip fragility. INTRODUCTION: Regional cortical thinning at the femoral neck is associated with hip fragility. Here, we investigated whether exercises involving high-magnitude impacts, moderate-magnitude impacts from odd directions, high-magnitude muscle forces, low-magnitude impacts at high repetition rate, or non-impact muscle forces at high repetition rate were associated with thicker femoral neck cortex. METHODS: Using three-dimensional magnetic resonance imaging, we scanned the proximal femur of 91 female athletes, representing the above-mentioned five exercise-loadings, and 20 referents. Cortical thickness at the inferior, anterior, superior, and posterior regions of the femoral neck was evaluated. Between-group differences were analyzed with ANCOVA. RESULTS: For the inferior cortical thickness, only the high-impact group differed significantly (approximately 60%, p = 0.012) from the reference group, while for the anterior cortex, both the high-impact and odd-impact groups differed (approximately 20%, p = 0.042 and p = 0.044, respectively). Also, the posterior cortex was approximately 20% thicker (p = 0.014 and p = 0.006, respectively) in these two groups. CONCLUSIONS: Odd-impact exercise-loading was associated, similar to high-impact exercise-loading, with approximately 20% thicker cortex around the femoral neck. Since odd-impact exercises are mechanically less demanding to the body than high-impact exercises, it is argued that this type of bone training would offer a feasible basis for targeted exercise-based prevention of hip fragility.

Augmenting Soft Tissue Contrast Using Edge-Enhancing Phase-Imaging Techniques in X-Ray Microtomography.

Conventional X-ray imaging is based on the attenuation of X-rays and the technique provides sufficient contrast when the difference between attenuation coefficients of neighboring structures is sufficient. A promising imaging possibility on a µCT is the use of phase information of an X-ray beam to generate an image of the sample. This is known as phase-contrast imaging. Propagation-based phase imaging sets the least amount of requirements on the imaging setup - lateral coherence for the X-ray source and a movable detector and source. The Zeiss Xradia MicroXCT-400 at our laboratory provides this possibility. Therefore, the phase-contrast imaging protocol, which provides an edge-enhancement effect, on the µCT device was optimized using thin polylactic acid fibers in order to enhance the visibility of low density samples. The optimization consisted of source and detector distance variation measurements. To demonstrate the contrast enhancement results, the optimization was applied to two types of collagen samples embedded in air, ethanol, and water.The results showed enhanced contrast for the edge-enhanced phase-contrast images compared to absorption images. Most importantly, the results indicated that the source does not need to placed at the negative limit to obtain useful phase information. Additionally, the visibility increases with increasing sample-to-detector distance. Finally, significantly enhanced contrast was obtained for the collagen sample embedded in water using phase-imaging techniques. The technique is limited due to the focal spot size and voltage of the X-ray source. The phase-imaging technique has the possibility to enhance contrast of low density samples and to reveal structures that cannot be seen using other imaging techniques.

Occurrence and species level diagnostics of Campylobacter spp., enteric Helicobacter spp. and Anaerobiospirillum spp. in healthy and diarrheic dogs and cats.

In order to study the occurrence and co-infection of different species of Campylobacter, enteric Helicobacter and Anaerobiospirillum in dogs and cats and define a possible association between these microrganisms and gastrointestinal disorders, 190 dogs and 84 cats, either healthy or with diarrhea, were sampled between 2002 and 2003. Thirty-three C. upsaliensis, 17 C. jejuni, 2 C. helveticus, 1 C. lari isolates from dogs and 14 C. helveticus, 7 C. jejuni, 6 C. upsaliensis isolates from cats were identified using species-specific PCR and phenotypic tests. Whole cell protein profile analysis, phenotypic tests, PCR-RFLP of gyrB and a phylogenetic study of partial groEL and 16S rRNA sequences were used to identify 37 H. bilis, 22 H. canis and 14 H. cinaedi in dogs and 12 H. canis, 5 H. bilis and 2 H. cinaedi in cats. Whole cell protein profile analysis, phenotypic tests and species-specific PCR of 16S rRNA were used to identify 14 A. succiniciproducens, 12 A. thomasii isolates and one unidentified Anaerobiospirillum sp. isolate in dogs and 3 A. thomasii isolates in cats. Fifty-two animals (19%) were positive for the isolation of more than one genus. No significant statistical correlation was found between any isolates of Campylobacter, Helicobacter or Anaerobiospirillum spp. or the various co-infection rates, and the presence of diarrhea in either dogs or cats. Campylobacter isolates were also tested for antibiotic resistance using the agar dilution method.

The influence of CSF on EEG sensitivity distributions of multilayered head models.

We examined how the cerebrospinal fluid (CSF) affects the distribution of electroencephalogram (EEG) measurement sensitivity. We used concentric spheres and realistic head models to investigate the difference between computed-tomography (CT) and magnetic resonance image (MRI) models that exclude the CSF layer. The cortical EEG sensitivity distributions support these phenomena and show that the CSF layer significantly influences them, thus identifying the importance of including the CSF layer inside the head volume conductor models. The results show that the highly conductive CSF channels the current, thus decreasing the maximum cortical current density relative to models that do not include the CSF. We found that the MRI and CT models yielded HSV results 20% and 45%, respectively, too small when compared with CSF-inclusive models.

Electrode position optimization for facial EMG measurements for human-computer interface.

OBJECTIVES: The aim of this work was to model facial electromyography (fEMG) to find optimal electrode positions for wearable wireless human-computer interface. The measurement system is a head cap developed in our institute and with it we can measure fEMG and electro-oculogram (EOG). The signals could be used to control the computer interface: gaze directions move the cursor and muscle activations correspond to clicking. METHODS: A very accurate 3D model of the human head was developed and it was used in the modeling of fEMG. The optimal positions of four electrodes on the forehead measuring the activations of frontalis and corrugator muscles were defined. Calculations were based on reciprocity theorem and lead field concept. RESULTS: A new accurate model is now in our use for modeling purposes. It has high spatial accuracy and number of inhomogeneities providing a good platform for various simulations. The best measurement sensitivity is achieved by placing the electrodes parallel to the muscle cells. Anyway, better separating capability for frontalis and corrugator activation is achieved by placing the electrodes more orthogonally. CONCLUSIONS: The developed model and the tools utilized are powerful methods to optimize the electrode positions of a wearable gaze and EMG-based user interface system. The modeling results provide direct feedback for developing next generation wearable head cap with optimized electrode locations.

Crystallization and sintering of borosilicate bioactive glasses for application in tissue engineering.

Typical silicate bioactive glasses are known to crystallize readily during the processing of porous scaffolds. While such crystallization does not fully suppress the bioactivity, the presence of significantly large amounts of crystals leads to a decrease in the rate of reaction of the glass and an uncontrolled release of ions. Furthermore, due to the non-congruent dissolution of silicate glasses, these materials have been shown to remain within the surgical site even 14 years post-operation. Therefore, bioactive materials that can dissolve more effectively and have higher conversion rates are required. Within this work, boron was introduced, in the FDA approved S53P4 glass, at the expense of SiO2. The crystallization and sintering-ability of the newly developed glasses were investigated by differential thermal analysis. All the glasses were found to crystallize primarily from the surface, and the crystal phase precipitation was dependent on the quantity of B2O3 incorporated. The rate of crystallization was found to be lower for the glasses when 25, 50 and 75% of SiO2 was replaced with B2O3. These glasses were further sintered into porous scaffolds using simple heat sintering. The impact of glass particle size and heat treatment temperature on the scaffold porosity and average pore size was investigated. Scaffolds with porosity ranging from 10 to 60% and compressive strength ranging from 1 to 35 MPa were produced. The scaffolds remained amorphous during processing and their ability to rapidly precipitate hydroxycarbonate apatite was maintained. This is of particular interest in the field of tissue engineering as scaffold degradation and reaction is generally faster and offers higher controllability as opposed to the current partially/fully crystallized scaffolds obtained from the FDA approved bioactive glasses.

Prolyl oligopeptidase colocalizes with α-synuclein, ß-amyloid, tau protein and astroglia in the post-mortem brain samples with Parkinson's and Alzheimer's diseases.

Prolyl oligopeptidase (EC 3.4.21.26, PREP) is a serine protease that hydrolyzes proline-containing peptides shorter than 30-mer but it has also nonhydrolytic functions. PREP has been shown to accelerate aggregation of wild-type α-synuclein (α-syn) under cell-free conditions, and PREP inhibitors can block this aggregation both in vitro and in vivo. α-syn is the main component of Lewy bodies in Parkinson's disease (PD) and Lewy body dementia. To clarify the possible interaction of PREP with other markers of neurodegenerative diseases, we studied colocalizations of PREP and (1) α-syn, (2) ß-amyloid, (3) tau protein and (4) astroglial and microglial cells in human post-mortem brain samples from PD, Alzheimer's disease (AD) patients and in healthy control brain samples. In the substantia nigra of PD brains, an intense colocalization with PREP and α-syn was evident. PREP colocalized also with ß-amyloid plaques in AD brains and with tau protein in AD and in healthy brains. PREP was also found in astroglial cells in PD, AD and control brains, but not in the microglia. Our findings are the first ones to demonstrate colocalization of PREP and pathological proteins in the human brain and support the view that, at least in spatial terms, PREP could be associated with pathogenesis of neurodegenerative diseases.

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease.

BACKGROUND AND PURPOSE: The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes.

Software suite for finite difference method models.

We have developed a software suite for finite difference method (FDM) model construction, visualization and quasi-static simulation to be used in bioelectric field modeling. The aim of the software is to provide a full path from medical image data to simulation of bioelectric phenomena and results visualization. It is written in Java and can be run on various platforms while still supporting all features included. The software can be distributed across a network utilizing dedicated servers for calculation intensive tasks. Supported visualization modes are both two- and three-dimensional modes.