Petro Julkunen

Depth-wise progression of osteoarthritis in human articular cartilage: investigation of composition, structure and biomechanics

OBJECTIVE Osteoarthritis (OA) is characterized by the changes in structure and composition of articular cartilage. However, it is not fully known, what is the depth-wise change in two major components of the cartilage solid matrix, i.e., collagen and proteoglycans (PGs), during OA progression. Further, it is unknown how the depth-wise changes affect local tissue strains during compression. Our aim was to address these issues. METHODS Data from the previous microscopic and biochemical measurements of the collagen content, distribution and orientation, PG content and distribution, water content and histological grade of normal and degenerated human patellar articular cartilage (n=73) were reanalyzed in a depth-wise manner. Using this information, a composition-based finite element (FE) model was used to estimate tissue function solely based on its composition and structure. RESULTS The orientation angle of collagen fibrils in the superficial zone of cartilage was significantly less parallel to the surface (P<0.05) in samples with early degeneration than in healthy samples. Similarly, PG content was reduced in the superficial zone in early OA (P<0.05). However, collagen content decreased significantly only at the advanced stage of OA (P<0.05). The composition-based FE model showed that under a constant stress, local tissue strains increased as OA progressed. CONCLUSION For the first time, depth-wise point-by-point statistical comparisons of structure and composition of human articular cartilage were conducted. The present results indicated that early OA is primarily characterized by the changes in collagen orientation and PG content in the superficial zone, while collagen content does not change until OA has progressed to its late stage. Our simulation results suggest that impact loads in OA joint could create a risk for tissue failure and cell death.

Effect of superficial collagen patterns and fibrillation of femoral articular cartilage on knee joint mechanics—A 3D finite element analysis

Collagen fibrils of articular cartilage have specific depth-dependent orientations and the fibrils bend in the cartilage surface to exhibit split-lines. Fibrillation of superficial collagen takes place in osteoarthritis. We aimed to investigate the effect of superficial collagen fibril patterns and collagen fibrillation of cartilage on stresses and strains within a knee joint. A 3D finite element model of a knee joint with cartilage and menisci was constructed based on magnetic resonance imaging. The fibril-reinforced poroviscoelastic material properties with depth-dependent collagen orientations and split-line patterns were included in the model. The effects of joint loading on stresses and strains in cartilage with various split-line patterns and medial collagen fibrillation were simulated under axial impact loading of 1000 N. In the model, the collagen fibrils resisted strains along the split-line directions. This increased also stresses along the split-lines. On the contrary, contact and pore pressures were not affected by split-line patterns. Simulated medial osteoarthritis increased tissue strains in both medial and lateral femoral condyles, and contact and pore pressures in the lateral femoral condyle. This study highlights the importance of the collagen fibril organization, especially that indicated by split-line patterns, for the weight-bearing properties of articular cartilage. Osteoarthritic changes of cartilage in the medial femoral condyle created a possible failure point in the lateral femoral condyle. This study provides further evidence on the importance of the collagen fibril organization for the optimal function of articular cartilage.

Factors influencing cortical silent period: Optimized stimulus location, intensity and muscle contraction

Inhibitory silent period (SP) is a transient suppression of voluntary muscle activity after depolarization of representative motor neuronal populations following transcranial magnetic stimulation (TMS). Our aim was to evaluate and present an optimal protocol for the measurement of SP by (1) determining the impact of muscle activation level and stimulus intensity (SI) on the duration of SP, and, (2) studying the relationship between motor evoked potential (MEP) and SP, using targeted stimulus delivery. Single magnetic pulses were focused on the optimal representation area of the thenar musculature on primary motor cortex. We utilized real-time 3D-positioning of TMS-evoked electric field on anatomical structures derived from individual MR-images. The SI varied from 80% to 120% of individual resting motor threshold (MT). Muscle activation levels varied from 20% to 80% of the maximal voluntary contraction (MVC). Contralateral SP lengthened significantly with increasing SI independent of target muscle activation. The peak amplitude of the MEP was affected by SI and force. Latency and duration of the MEP were practically unaffected by SI or force. Focal stimulation at 110-120% MT and approximately 50% MVC (with only negligible need for control) provides most stable and informative SP. MEP should be included in SP as the error from marking the onset diminishes. This study provides a guideline for the consistent measurement of SP, which is applicable when using navigated or traditional TMS.

Stress–relaxation of human patellar articular cartilage in unconfined compression: Prediction of mechanical response by tissue composition and structure

Mechanical properties of articular cartilage are controlled by tissue composition and structure. Cartilage function is sensitively altered during tissue degeneration, in osteoarthritis (OA). However, mechanical properties of the tissue cannot be determined non-invasively. In the present study, we evaluate the feasibility to predict, without mechanical testing, the stress-relaxation response of human articular cartilage under unconfined compression. This is carried out by combining microscopic and biochemical analyses with composition-based mathematical modeling. Cartilage samples from five cadaver patellae were mechanically tested under unconfined compression. Depth-dependent collagen content and fibril orientation, as well as proteoglycan and water content were derived by combining Fourier transform infrared imaging, biochemical analyses and polarized light microscopy. Finite element models were constructed for each sample in unconfined compression geometry. First, composition-based fibril-reinforced poroviscoelastic swelling models, including composition and structure obtained from microscopical and biochemical analyses were fitted to experimental stress-relaxation responses of three samples. Subsequently, optimized values of model constants, as well as compositional and structural parameters were implemented in the models of two additional samples to validate the optimization. Theoretical stress-relaxation curves agreed with the experimental tests (R=0.95-0.99). Using the optimized values of mechanical parameters, as well as composition and structure of additional samples, we were able to predict their mechanical behavior in unconfined compression, without mechanical testing (R=0.98). Our results suggest that specific information on tissue composition and structure might enable assessment of cartilage mechanics without mechanical testing.

Biomechanical, biochemical and structural correlations in immature and mature rabbit articular cartilage.

OBJECTIVE The structure and composition of articular cartilage change during development and growth. These changes lead to alterations in the mechanical properties of cartilage. In the present study, biomechanical, biochemical and structural relationships of articular cartilage during growth and maturation of rabbits are investigated. DESIGN Articular cartilage specimens from the tibial medial plateaus and femoral medial condyles of female New Zealand white rabbits were collected from seven age-groups; 0 days (n=29), 11 days (n=30), 4 weeks (n=30), 6 weeks (n=30), 3 months (n=24), 6 months (n=24) and 18 months (n=19). The samples underwent mechanical testing under creep indentation. From the mechanical response, instantaneous and equilibrium moduli were determined. Biochemical analyses of tissue collagen, hydroxylysylpyridinoline (HP) and pentosidine (PEN) cross-links in full thickness cartilage samples were conducted. Proteoglycans were investigated depth-wise from the tissue sections by measuring the optical density of Safranin-O-stained samples. Furthermore, depth-wise collagen architecture of articular cartilage was analyzed with polarized light microscopy. Finite element analyses of the samples from different age-groups were conducted to reveal tensile and compressive properties of the fibril network and the matrix of articular cartilage, respectively. RESULTS Tissue thickness decreased from approximately 3 to approximately 0.5mm until the age of 3 months, while the instantaneous modulus increased with age prior to peak at 4-6 weeks. A lower equilibrium modulus was observed before 3-month-age, after which the equilibrium modulus continued to increase. Collagen fibril orientation angle and parallelism index were inversely related to the instantaneous modulus, tensile fibril modulus and tissue thickness. Collagen content and cross-linking were positively related to the equilibrium compressive properties of the tissue. CONCLUSIONS During maturation, significant modulation of tissue structure, composition and mechanical properties takes place. Importantly, the present study provides insight into the mechanical, chemical and structural interactions that lead to functional properties of mature articular cartilage.

Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes.

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints. Understanding the detailed effects of the mechanical environment of chondrocytes on cell behavior is therefore essential for the study of the development, adaptation, and degeneration of articular cartilage. Recent progress in macroscopic models has improved our understanding of depth-dependent properties of cartilage. However, none of the previous works considered the effect of realistic collagen orientation or depth-dependent negative charges in microscopic models of chondrocyte mechanics. The aim of this study was to investigate the effects of the collagen network and fixed charge densities of cartilage on the mechanical environment of the chondrocytes in a depth-dependent manner. We developed an anisotropic, inhomogeneous, microstructural fibril-reinforced finite element model of articular cartilage for application in unconfined compression. The model consisted of the extracellular matrix and chondrocytes located in the superficial, middle, and deep zones. Chondrocytes were surrounded by a pericellular matrix and were assumed spherical prior to tissue swelling and load application. Material properties of the chondrocytes, pericellular matrix, and extracellular matrix were obtained from the literature. The loading protocol included a free swelling step followed by a stress-relaxation step. Results from traditional isotropic and transversely isotropic biphasic models were used for comparison with predictions from the current model. In the superficial zone, cell shapes changed from rounded to elliptic after free swelling. The stresses and strains as well as fluid flow in cells were greatly affected by the modulus of the collagen network. The fixed charge density of the chondrocytes, pericellular matrix, and extracellular matrix primarily affected the aspect ratios (height/width) and the solid matrix stresses of cells. The mechanical responses of the cells were strongly location and time dependent. The current model highlights that the collagen orientation and the depth-dependent negative fixed charge densities of articular cartilage have a great effect in modulating the mechanical environment in the vicinity of chondrocytes, and it provides an important improvement over earlier models in describing the possible pathways from loading of articular cartilage to the mechanical and biological responses of chondrocytes.

Motor potentials evoked by navigated transcranial magnetic stimulation in healthy subjects.

Summary: Navigated transcranial magnetic stimulation (TMS) is a tool for targeted, noninvasive stimulation of cerebral cortex. Transcranial stimuli can depolarize neurons and evoke measurable effects which are unique in two ways: the effects are caused directly and without a consciousness of the subject, and, the responses from peripheral muscles provide a direct measure for the integrity of the whole motor pathway. The clinical relevance of the method has not always been fully exposed because localizing the optimal stimulation site and determining the optimal stimulation strength have been dependent on time-consuming experimentation and skill. Moreover, in many disorders it has been uncertain, whether the lack of motor responses is the result of true pathophysiological changes or merely because of unoptimal stimulation. We characterized the muscle responses from human primary motor cortex system by navigated TMS to provide normative values for the clinically relevant TMS parameters on 65 healthy volunteers aged 22 to 81 years. We delivered focal TMS pulses on the primary motor area (M1) and recorded muscle responses on thenar and anterior tibial muscles. Motor threshold, latencies and amplitudes of motor-evoked potentials, and silent period duration were measured. The correction of the motor-evoked potential latency for subjects’ height is provided. In conclusion, we provide a modified baseline of TMS-related parameters for healthy subjects. Earlier such large-scale baseline material has not been available.

Precision of nanoindentation protocols for measurement of viscoelasticity in cortical and trabecular bone

Nanoindentation has recently gained attention as a characterization technique for mechanical properties of biological tissues, such as bone, on the sub-micron level. However, optimal methods to characterize viscoelastic properties of bones are yet to be established. This study aimed to compare the time-dependent viscoelastic properties of bone tissue obtained with different nanoindentation methods. Bovine cortical and trabecular bone samples (n=8) from the distal femur and proximal tibia were dehydrated, embedded and polished. The material properties determined using nanoindentation were hardness and reduced modulus, as well as time-dependent parameters based on creep, loading-rate, dissipated energy and semi-dynamic testing under load control. Each loading protocol was repeated 160 times and the reproducibility was assessed based on the coefficient of variation (CV). Additionally, three well-characterized polymers were tested and CV values were calculated for reference. The employed methods were able to characterize time-dependent viscoelastic properties of bone. However, their reproducibility varied highly (CV 9-40%). The creep constant increased with increasing dwell time. The reproducibility was best with a 30s creep period (CV 18%). The dissipated energy was stable after three repeated load cycles, and the reproducibility improved with each cycle (CV 23%). The viscoelastic properties determined with semi-dynamic test increased with increase in frequency. These measurements were most reproducible at high frequencies (CV 9-10%). Our results indicate that several methods are feasible for the determination of viscoelastic properties of bone material. The high frequency semi-dynamic test showed the highest precision within the tested nanoindentation protocols.

Navigated TMS combined with EEG in mild cognitive impairment and Alzheimer's disease: a pilot study.

Our aim was to assess the potential of navigated transcranial magnetic stimulation (TMS)-evoked electroencephalographic (EEG) responses in studying neuronal reactivity and cortical connectivity in Alzheimers disease (AD) and in mild cognitive impairment (MCI). We studied 14 right-handed subjects: five patients with AD, five patients with MCI and four healthy controls. Fifty TMS-pulses at an intensity of 110% of individually determined motor threshold were delivered to the hand area of primary motor cortex (M1) with navigated brain stimulation (NBS). Spreading of primary NBS-evoked neuronal activity was monitored with a compatible 60-channel EEG, and analyzed in time, frequency and spatial-domains. We found significantly reduced TMS-evoked P30 (time-locked response 30 ms after the magnetic stimulation) in the AD subjects. This reduction was seen in the temporo-parietal area ipsilateral to stimulation side as well as in the contralateral fronto-central cortex corresponding to the sensorimotor network, which is anatomically interconnected with the stimulated M1. In addition, there was a significant decrease in the N100 amplitude in the MCI subjects when compared with the control subjects. Thus, the combination of NBS and EEG revealed prominent changes in functional cortical connectivity and reactivity in the AD subjects. This pilot study suggests that the method may provide a novel tool for examining the degree and progression of dementia.

Navigated transcranial magnetic stimulation and computed electric field strength reduce stimulator-dependent differences in the motor threshold

The motor threshold (MT) is a fundamental parameter for evaluating cortical excitability in transcranial magnetic stimulation (TMS) despite remarkable variation, both within, and between subjects. We intended to test whether the variation could be reduced by targeting the stimulation on-line and modeling the TMS-induced electric field on individual MR images. Navigated TMS was used to map the primary motor cortex for the representation area of the thenar muscles (abductor pollicis brevis) and to determine the MT. Thirteen healthy subjects participated in the study. To determine the between-subject variation, the MTs of nine subjects were measured with two different stimulators (comparison study). To study the individual variation, the MT measurement was repeated 20 times in four subjects always using the same stimulator (longitudinal study). In the comparison study, the MTs differed significantly between the two stimulators over all subjects (p<0.001), whereas the electric field strengths did not exhibit significant difference between the stimulators. Both, the MTs, and the electric field strengths showed similar variations, which were greater between subjects (comparison study) than within subjects (longitudinal study). In the comparison study, the distance between the locations of the two different coils on the scalp was significantly greater than the distance between the induced electric field maxima in the brain (p<0.001). We conclude that on-line navigation can be used to reduce the variation caused by different stimulator types and individual subject anatomy. In addition, cortical excitability can be evaluated by using computed electric field strength as well as stimulator-dependent MT.