Early Detection of Cartilage Degeneration: A Comparison of Histology, Fiber Bragg Grating-Based Micro-Indentation, and Atomic Force Microscopy-Based Nano-Indentation.

We have determined the sensitivity and detection limit of a new fiber Bragg grating (FBG)-based optoelectronic micro-indenter for biomechanical testing of cartilage and compared the results to indentation-type atomic force microscopy (IT-AFM) and histological staining. As test samples, we used bovine articular cartilage, which was enzymatically degraded ex vivo for five minutes using different concentrations of collagenase (5, 50, 100 and 500 µg/mL) to mimic moderate extracellular matrix deterioration seen in early-stage osteoarthritis (OA). Picrosirius Red staining and polarization microscopy demonstrated gradual, concentration-dependent disorganization of the collagen fibrillar network in the superficial zone of the explants. Osteoarthritis Research Society International (OARSI) grading of histopathological changes did not discriminate between undigested and enzymatically degraded explants. IT-AFM was the most sensitive method for detecting minute changes in cartilage biomechanics induced by the lowest collagenase concentration, however, it did not distinguish different levels of cartilage degeneration for collagenase concentrations higher than 5 µg/mL. The FBG micro-indenter provided a better and more precise assessment of the level of cartilage degeneration than the OARSI histological grading system but it was less sensitive at detecting mechanical changes than IT-AFM. The FBG-sensor allowed us to observe differences in cartilage biomechanics for collagenase concentrations of 100 and 500 µg/mL. Our results confirm that the FBG sensor is capable of detecting small changes in articular cartilage stiffness, which may be associated with initial cartilage degeneration caused by early OA.

Mice Lacking the Matrilin Family of Extracellular Matrix Proteins Develop Mild Skeletal Abnormalities and Are Susceptible to Age-Associated Osteoarthritis.

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4-/- mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4-/- mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4-/- mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4-/- mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

Adipose-derived stem/progenitor cells from lipoaspirates: A comparison between the Lipivage200-5 liposuction system and the Body-Jet liposuction system.

BACKGROUND: Adipose-derived stem/progenitor cells (ADSPCs) are under investigation in many clinical applications for their regenerative potential in a variety of autoimmune, degenerative, and inflammatory diseases. Adipose tissue, which is mainly harvested by manual liposuction, is the main source of these ADSPCs. OBJECTIVE: In the past years, a variety of different liposuction devices have been commercialized. To ensure a high quality of obtained ADSPCs, it is crucial to show the advantages and disadvantages of frequently used liposuction methods. For this reason, the objective of this study was to compare ADSPCs harvested by either the suction-assisted LipiVage200-5 or the water-assisted Body-Jet system. METHODS: The proliferation potential of ADSPCs, harvested from 20 patients, was assessed by cumulative population doublings (cumPD), population doubling time (PDT), colony-forming units (CFU), and cell metabolism assays. To prove the multipotency of the primary isolated cells, ADSPCs were induced to differentiate into adipogenic, osteogenic, and chondrogenic lineages. RESULTS: Our data show a significantly higher cumPD, as well as a significantly lower PDT for cells obtained by the Body-Jet system. No significant differences were found regarding the CFU efficiency and the cell metabolism. Furthermore, we showed that the adipogenic, osteogenic, and chondrogenic potential of ADSPCs is similar in both groups. DISCUSSION/CONCLUSION: In our study, we provide evidence that the cell characteristics and the functional properties of ADSPCs isolated after liposuction with different techniques are largely similar. However, we observed a significantly higher cumPD and a slightly higher adipogenic potential in cells isolated after liposuction with the Body-Jet system. Different cannula sizes and sheer stresses in the used methods might play a role here.

Lamotrigine Induces Tremor among Epilepsy Patients Probably via Cerebellar Pathways.

Lamotrigine, a frequently used antiepileptic drug, inhibits voltage-gated sodium-channels. By suppressing the release of glutamate and aspartate, lamotrigine acts as a membrane stabilizer, and it is also effective in bipolar disorder and migraine. However, lamotrigine is known to induce tremor among 4-10% of patients. We examined the lamotrigine-induced tremor in 28 epilepsy patients (age: 38.06 ± 13.56 years; 24 females and 4 males) receiving lamotrigine monotherapy and compared the data to 30 age- and sex-matched controls (age: 33.06 ± 10.71 years; 25 females and 5 males). Tremor was visually assessed by clinical tremor rating scales. Quantitative characteristics (intensity, center frequency and frequency dispersion) which are regularly used to differentiate various tremor syndromes were measured by validated, sensitive biaxial accelerometry in resting, postural and intentional positions. Regularity of repetitive finger and hand movements and reaction time were also determined. Data were statistically analyzed. Clinical tremor rating scales detected pathological tremor in three patients (10%), while accelerometry revealed tremor in seven patients (25%). Center frequency of patients with pathological tremor was similar to controls, but the frequency dispersion was significantly lower and tremor intensity was significantly higher in both postural and intentional positions. Rhythmic movements and reaction time were normal. Our results show that objective measurements detect pathological intention tremor in 25% of epilepsy patients receiving lamotrigine monotherapy. Quantitative characteristics suggest the involvement of the cerebellum in the pathomechanism of lamotrigine-induced tremor. Determining the parameters of drug-induced tremor syndromes might help to understand the complex action of tremor generator networks.

Aggrecan Hypomorphism Compromises Articular Cartilage Biomechanical Properties and Is Associated with Increased Incidence of Spontaneous Osteoarthritis.

The gene encoding the proteoglycan aggrecan (Agc1) is abundantly expressed in cartilage during development and adulthood, and the loss or diminished deposition of the protein results in a wide range of skeletal malformations. Furthermore, aggrecan degradation is a hallmark of cartilage degeneration occurring in osteoarthritis. In the present study, we investigated the consequences of a partial loss of aggrecan in the postnatal skeleton and in the articular cartilage of adult mice. We took advantage of the previously described Agc1tm(IRES-CreERT2) mouse line, which allows for conditional and timely-regulated deletion of floxed, cartilage-expressed genes. As previously reported, the introduction of the CreERT2 cassette in the 3'UTR causes a disruption of the normal expression of Agc1 resulting in a hypomorphic deposition of the protein. In homozygous mice, we observed a dwarf phenotype, which persisted throughout adulthood supporting the evidence that reduced aggrecan amount impairs skeletal growth. Homozygous mice exhibited reduced proteoglycan staining of the articular cartilage at 6 and 12 months of age, increased stiffening of the extracellular matrix at six months, and developed severe cartilage erosion by 12 months. The osteoarthritis in the hypomorph mice was not accompanied by increased expression of catabolic enzymes and matrix degradation neoepitopes. These findings suggest that the degeneration found in homozygous mice is likely due to the compromised mechanical properties of the cartilage tissue upon aggrecan reduction.

Impact of vitamin E-blended UHMWPE wear particles on the osseous microenvironment in polyethylene particle-induced osteolysis.

Aseptic loosening mediated by wear particle-induced osteolysis (PIO) remains the major cause of implant loosening in endoprosthetic surgery. The development of new vitamin E (α-tocopherol)-blended ultra-high molecular weight polyethylene (VE-UHMWPE) with increased oxidation resistance and improved mechanical properties has raised hopes. Furthermore, regenerative approaches may be opened, as vitamin E supplementation has shown neuroprotective characteristics mediated via calcitonin gene-related peptide (CGRP), which is known to affect bone remodeling in PIO. Therefore, the present study aimed to further clarify the impact of VE-UHMWPE wear particles on the osseous microenvironment and to identify the potential modulatory pathways involved. Using an established murine calvaria model, mice were subjected to sham operation (SHAM group), or treated with UHMWPE or VE-UHMWPE particles for different experimental durations (7, 14 and 28 days; n=6/group). Morphometric analysis by micro-computed tomography detected significant (p<0.01) and comparable signs of PIO in all particle-treated groups, whereas markers of inflammation [tumor necrosis factor (TNF)-α/tartrate resistant acid phosphatase (TRAP) staining] and bone remodeling [Dickkopf-related protein 1 (DKK-1)/osteoprotegerin (OPG)] were most affected in the early stages following surgery. Taking the present data into account, VE-UHMWPE appears to have a promising biocompatibility and increased ageing resistance. According to the α-CGRP serum levels and immunohistochemistry, the impact of vitamin E on neuropeptidergic signaling and its chance for regenerative approaches requires further investigation.

[Cerebral manifestations of thromboangiitis obliterans. Case report]. / Thromboangitis obliterans agyi manifesztációja.

Thromboangiits obliterans (Buerger's disease) is a non-atherosclerotic, segmental inflammatory and obliterative disease affecting small and medium sized arteries and veins. The etiology is still unknown, but it is in close relationship with tobacco use. Symptoms begin under the age of 45 years and the undulating course is typical. Patients usually present with acute and chronic ischemic or infectious acral lesions. Diagnosis is usually based on clinical and angiographic criteria and it is important to exclude autoimmune disease, thrombophilia, diabetes, and proximal embolic sources. Even though Buerger's disease most commonly involves the arteries of the extremities, the pathologic findings sometimes affect the cerebral, coronary and internal thoracic, renal and mesenteric arteries as well. The authors present the history of a patient with known Buerger's disease and acute ischemic stroke. Brain imaging detected acute and chronic ischemic lesions caused by middle cerebral non-atherosclerotic arteriopathy on the symptomatic side. Other etiology was excluded by detailed investigations. Orv. Hetil., 2016, 157(30), 1207-1211.

Structural and mechanical properties of the proliferative zone of the developing murine growth plate cartilage assessed by atomic force microscopy.

The growth plate (GP) is a dynamic tissue driving bone elongation through chondrocyte proliferation, hypertrophy and matrix production. The extracellular matrix (ECM) is the major determinant of GP biomechanical properties and assumed to play a pivotal role for chondrocyte geometry and arrangement, thereby guiding proper growth plate morphogenesis and bone elongation. To elucidate the relationship between morphology and biomechanics during cartilage morphogenesis, we have investigated age-dependent structural and elastic properties of the proliferative zone of the murine GP by atomic force microscopy (AFM) from the embryonic stage to adulthood. We observed a progressive cell flattening and arrangement into columns from embryonic day 13.5 until postnatal week 2, correlating with an increasing collagen density and ECM stiffness, followed by a nearly constant cell shape, collagen density and ECM stiffness from week 2 to 4 months. At all ages, we found marked differences in the density and organization of the collagen network between the intracolumnar matrix, and the intercolumnar matrix, associated with a roughly two-fold higher stiffness of the intracolumnar matrix compared to the intercolumnar matrix. This difference in local ECM stiffness may force the cells to arrange in a columnar structure upon cell division and drive bone elongation during embryonic and juvenile development.

Early changes in morphology, bone mineral density and matrix composition of vertebrae lead to disc degeneration in aged collagen IX -/- mice.

Collagen IX (Col IX) is an important component of the cartilage extracellularmatrix and has been associated with degenerative cartilage disorders and chondrodysplasias in humans. Further, polymorphisms in Col IX are known risk factors for the development of early intervertebral disc (IVD) degeneration. To understand the role of Col IX in the pathogenesis of IVD disorders, the spine of newborn and older Col IX deficient mice was systematically analyzed and compared to C57BL/6N controls. Morphology and bone parameters of the spine from newborn, 6 and 10 months old animals were investigated using µCT measurements. Histological staining was used to evaluate tissue structure and degree of degeneration. Localization and expression of extracellularmatrix proteins was analyzed in depth by immunofluorescence staining, immunoblotting, RT-PCR and in situ hybridization. High resolution imaging and stiffness measurements were performed by atomic force microscopy (AFM). Vertebral bodies of newborn Col IX-deficient mice were smaller and showed an increased mineral density compared to wild type animals. At birth, lack of Col IX led to a disrupted cellular organization in the cartilaginous endplate and a smaller nucleus pulposus of the IVD.Expression levels and localization of other extracellularmatrix proteins were strongly altered accompanied by a softening of cartilaginous tissues. In older animals, absence of Col IX caused earlier and more pronounced disc degeneration with annular fissures. The absence of Col IX induces early developmental, structural and biomechanical alterations in both vertebral body and intervertebral disc which eventually cause severe degenerative changes in the aging spine.

Muscle contraction controls skeletal morphogenesis through regulation of chondrocyte convergent extension.

Convergent extension driven by mediolateral intercalation of chondrocytes is a key process that contributes to skeletal growth and morphogenesis. While progress has been made in deciphering the molecular mechanism that underlies this process, the involvement of mechanical load exerted by muscle contraction in its regulation has not been studied. Using the zebrafish as a model system, we found abnormal pharyngeal cartilage morphology in both chemically and genetically paralyzed embryos, demonstrating the importance of muscle contraction for zebrafish skeletal development. The shortening of skeletal elements was accompanied by prominent changes in cell morphology and organization. While in control the cells were elongated, chondrocytes in paralyzed zebrafish were smaller and exhibited a more rounded shape, confirmed by a reduction in their length-to-width ratio. The typical columnar organization of cells was affected too, as chondrocytes in various skeletal elements exhibited abnormal stacking patterns, indicating aberrant intercalation. Finally, we demonstrate impaired chondrocyte intercalation in growth plates of muscle-less Sp(d) mouse embryos, implying the evolutionary conservation of muscle force regulation of this essential morphogenetic process.Our findings provide a new perspective on the regulatory interaction between muscle contraction and skeletal morphogenesis by uncovering the role of muscle-induced mechanical loads in regulating chondrocyte intercalation in two different vertebrate models.

Quantitative analysis of motor performance in epilepsy patients treated with valproate.

PURPOSE: The aim of our study was to detect objective signs of deterioration of motor performance in epilepsy patients treated with chronic valproate therapy. METHODS: We examined 14 controls and 15 epilepsy patients receiving chronic valproate monotherapy, who had no subjective complaints related to motor function. Regularity and maximum frequency of repetitive hand and finger movements, and simple reaction time were measured. Intensity and frequency characteristics of resting and postural tremor were assessed using accelerometry. Data were statistically evaluated. RESULTS: Repetitive hand and finger movements were significantly more irregular and the maximum frequency of repetitive movements was significantly lower in the valproate group than in controls. Resting tremor peak frequency and motor reaction time of the two cohorts did not differ. CONCLUSIONS: This is the first study, which quantitatively assesses motor performance of patients with epilepsy on chronic valproate therapy. The results suggest significant irregularity of repetitive hand movements and finger tapping even in patients with no motor complaints. Objective methods might help to recognize valproate-induced motor performance deterioration.

Impaired rhythm generation in essential tremor.

It has been suggested that the cerebellum plays a role in the event-based timing of synchronized repetitive movements. We hypothesized that regularity of rhythmic movements in essential tremor (ET) is impaired, since several lines of evidence suggest the involvement of the cerebellum in the pathomechanism of ET. To test this assumption, we examined the regularity and the maximum frequency of auditory paced repetitive movements at slow and fast stimulus rate in 34 ET patients. Variability of rhythmic finger tapping and alternating hand movements, defined by the standard deviation of movement offset before or after the pacing signal, was significantly higher compared to healthy controls. Timing of rhythmic movements of the two hands was disturbed to the same degree. Our results suggest a severe deficit of event-based rhythm generation on both sides in ET, supporting the presumed bilateral cerebellar dysfunction in this disorder.

[Complex tremor analysis for the differential diagnosis of essential tremor and Parkinson's disease]. / Komplex tremorometria az esszenciális tremor es a Parkinson-szindróma elkülönítésében.

OBJECTIVE: Tremor is the most common movement disorder which is most often either essential or caused by Parkinson's disease. The differentiation of these disorders at the initial stage may be difficult. Objective assessment of the efficacy of tremor medications is only possible by instrumental measurements. The aim of this study was to determine whether the computer assisted tremor analysis system CATSYS 2000 can help in the differentiation of parkinsonian from essential tremor. METHODS: The rhythmicity and maximal frequency of fast alternating hand and finger movements, simple reaction time and postural instability were recorded in healthy controls (n = 18), patients with Parkinson's disease (n = 39) and essential tremor (n = 37). Data were digitally converted and statistically analyzed. RESULTS: Tremor intensity, median frequency and frequency distribution showed characteristic differences in the three groups. Performance in fast alternating movements of hands and fingers were significantly worse in both tremor groups compared to the healthy controls. CONCLUSIONS: The data also indicated that quantitative measurements of tremor parameters must be performed on both sides, because the presence of significant side differences support the diagnosis of Parkinson's disease. The method presented can be used to objectively analyze tremor and performance in rhythmic movements. The results show that it helps to differentiate parkinsonian from essential tremor as well as to predict disease course and the effectiveness of therapy. Multivariate statistical analysis of tremor and movement performance also provides an opportunity to study the pathogenesis of human tremor.

Asymmetry of tremor intensity and frequency in Parkinson's disease and essential tremor.

We investigated the asymmetry of tremor intensity, frequency and frequency dispersion of Parkinsonian (PT) and essential (ET) tremor using accelerometry. Data of the more and less trembling hands were statistically elaborated. We found that tremor intensity was significantly asymmetric not only in PT but also in ET, while frequency and frequency dispersion were symmetric in ET but asymmetric in PT. We conclude that bilateral assessment of frequency related tremor parameters may be used for differentiation between ET and PT, and provides further details on the central organization of tremor generators.