Thermal Dynamics of a Novel Radio-Frequency Device for Endoscopic Spine Surgery: An Experimental Model.

STUDY DESIGN: Experimental study. OBJECTIVE: In this study, the ambient temperature of a radiofrequency (RF) electrode tip was compared and analyzed in terms of products, mode, flow quantity, and flow rate. SUMMARY OF BACKGROUND DATA: Endoscopic spine surgery is a widely used operation for degenerative lumbar stenosis and herniated lumbar disc. To perform endoscopic spine surgery, dedicated instruments like a RF generator and electrode are essential. METHODS: An evaluation system capable of measuring temperature under equal conditions at a certain distance from the electrode tip was manufactured. The distance between the electrode tip and the temperature sensor was set to 1, 5, and 10 mm. The flow quantities of 0, 50, 100, and 150 mL/min and the flow rates of 0, 0.20, 0.53, and 0.80 m/s were compared and statistically analyzed. RESULTS: The temperatures measured in the experiments conducted on the four combinations of RF device showed similar values, and showed differences according to the characteristics of each mode of the RF. As the distance between the electrode tip and the temperature sensor increased, the temperature decreased, and as flow quantity or flow rate increased, the temperature decreased. The maximum temperatures differed significantly according to flow quantity, between flow quantities of 0 and 100 mL/min (P  = 0.03) and between 0 and 150 mL/min (P ≤ 0.01). The maximum temperatures also differed significantly between the flow rate of 0 m/s, and the flow rates of 0.20, 0.53, and 0.80 m/s, with P ≤ 0.01 in all three comparisons. CONCLUSION: This is the first study in which we made a customized RF temperature evaluation system and verified the temperature changes in various environments. When irrigation was performed, we could confirm that the maximum temperature was less than 60°C. Irrigation is considered essential in endoscopic spine surgery. LEVEL OF EVIDENCE: 3.

Botox Injections in Paraspinal Muscles Result in Low Maximal Specific Force and Shortening Velocity in Fast but Not Slow Skinned Muscle Fibers.

STUDY DESIGN: Basic science, experimental animal study. OBJECTIVE: To determine the effects of Botulinum toxin type A (BTX-A) injections on the mechanical properties of skinned muscle fibers (cells) of rabbit paraspinal muscles. SUMMARY OF BACKGROUND DATA: BTX-A has been widely used in the treatment of disorders of muscle hyperactivity, such as spasticity, dystonia, and back pain. However, BTX-A injection has been shown to cause muscle atrophy, fat infiltration, and decreased force output in target muscles, but its potential effects on the contractile machinery and force production on the cellular level remain unknown. METHODS: Nineteen-month-old, male New Zealand White Rabbits received either saline or BTX-A injections into the paraspinal muscles, equally distributed along the left and right sides of the spine at T12, L1, and L2 at 0, 8, 12, 16, 20, and 24 weeks. Magnetic resonance imaging was used to quantify muscle crosssectional area and structural changes before and at 28 weeks following the initial injection. Skinned fibers isolated from the paraspinal muscles were tested for their active and passive force-length relationships, unloaded shortening velocity, and myosin heavy chain isoforms. RESULTS: BTX-A injections led to significant fat infiltration within the injected muscles and a greater proportion of IIa to IIx fibers. Isolated fast fibers from BTX-A injected animals had lower active force and unloaded shortening velocity compared with fibers from saline-injected control animals. Force and velocity properties were not different between groups for the slow fibers. CONCLUSION: Injection of BTX-A into the paraspinal rabbit muscles leads to significant alterations in the contractile properties of fast, but not slow, fibers.Level of Evidence: N/A.

Predicting Sarcopenia of Female Elderly from Physical Activity Performance Measurement Using Machine Learning Classifiers.

PURPOSE: Sarcopenia is a symptom in which muscle mass decreases due to decreasing in the number of muscle fibers and muscle cross-sectional area as aging. This study aimed to develop a machine learning classification model for predicting sarcopenia through a inertial measurement unit (IMU)-based physical performance measurement data of female elderly. PATIENTS AND METHODS: Seventy-eight female subjects from an elderly population (aged: 78.8±5.7 years) volunteered to participate in this study. To evaluate the physical performance of the elderly, the experiment conducted timed-up-and-go test (TUG) and 6-minute walk test (6mWT) with worn a single IMU. Based on literature review, 132 features were extracted from collected data. Feature selection was performed through the Kruskal-Wallis test, and features datasets were constructed according to feature selection. Three major machine learning-based classification algorithms classified the sarcopenia group in each dataset, and the performance of classification models was compared. RESULTS: As a result of comparing the classification model performance for sarcopenia prediction, the k-nearest neighborhood algorithm (kNN) classification model using 40 major features of TUG and 6mWT showed the best performance at 88%. CONCLUSION: This study can be used as a basic research for the development of self-monitoring technology for sarcopenia.

Three-dimensional micro-scale strain mapping in living biological soft tissues.

Non-invasive characterization of the mechanical micro-environment surrounding cells in biological tissues at multiple length scales is important for the understanding of the role of mechanics in regulating the biosynthesis and phenotype of cells. However, there is a lack of imaging methods that allow for characterization of the cell micro-environment in three-dimensional (3D) space. The aims of this study were (i) to develop a multi-photon laser microscopy protocol capable of imprinting 3D grid lines onto living tissue at a high spatial resolution, and (ii) to develop image processing software capable of analyzing the resulting microscopic images and performing high resolution 3D strain analyses. Using articular cartilage as the biological tissue of interest, we present a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning length scales from the tissue to the cell level. Using custom image processing software, we provide accurate and robust 3D micro-strain analysis that allows for detailed qualitative and quantitative assessment of the 3D tissue kinematics. This novel technique preserves tissue structural integrity post-scanning, therefore allowing for multiple strain measurements at different time points in the same specimen. The proposed technique is versatile and opens doors for experimental and theoretical investigations on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. STATEMENT OF SIGNIFICANCE: We presented a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning from tissue length scale to cellular length scale. Using a custom image processing software (lsmgridtrack), we provide accurate and robust micro-strain analysis that allowed for detailed qualitative and quantitative assessment of the 3D tissue kinematics. The approach presented here can also be applied to other biological tissues such as meniscus and annulus fibrosus, as well as tissue-engineered tissues for the characterization of their mechanical properties. This imaging technique opens doors for experimental and theoretical investigation on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues.

In vivo study of paraspinal muscle weakness using botulinum toxin in one primate model.

BACKGROUND: It has been generally speculated that paraspinal muscle weakness is related to the spinal degeneration including intervertebral disc failure. The purpose of this study was to investigate the effects of paraspinal muscle weakness induced by the botulinum toxin type-A on the lumbar spine and behavior pattern in an in-vivo primate model which has an upright locomotion similar to that of humans. METHODS: Botox injections into paraspinal muscle of one cynomolgus monkey were conducted biweekly up to 19 weeks at L2-L3, L3-L4 and L4-L5. MRIs were performed for measurement of muscle cross-sectional areas and behavioral data were collected using a high-resolution portable digital video camera. FINDINGS: The cross-sectional areas of the paraspinal muscles at L2-L3, L3-L4 and L4-L5 decreased by 8%, 12% and 8% at 21 weeks after the Botox injection, respectively. Intervertebral disc thickness at L2-L3, L3-L4 and L4-L5 decreased by 6%, 8% and 5% at 21 weeks after initial Botox injection, respectively. After the Botox injections, locomotion and movement activity of the monkey was decreased. The duration of sitting increased from 21% to a maximum of 97% at 9 weeks after the Botox injection, while stance time decreased from 9% to a minimum of 1% at 11 weeks post Botox injection. INTERPRETATION: The findings of this study revealed that paraspinal muscle atrophy affects intervertebral disc morphology and locomotion activity of a primate and may lead to an onset of intervertebral disc degeneration.

Alterations in structural macromolecules and chondrocyte deformations in lapine retropatellar cartilage 9 weeks after anterior cruciate ligament transection.

The structural integrity and mechanical environment of the articular cartilage matrix directly affect chondrocyte deformations. Rabbit models of early osteoarthritis at 9 weeks following anterior cruciate ligament transection (ACLT) have been shown to alter the deformation behavior of superficial zone chondrocytes in mechanically loaded articular cartilage. However, it is not fully understood whether these changes in cell mechanics are caused by changes in structural macromolecules in the extracellular matrix. Therefore, the purpose of this study was to characterize the proteoglycan content, collagen content, and collagen orientation at 9 weeks post ACLT using microscopic techniques, and relate these changes to the altered cell mechanics observed upon mechanical loading of cartilage. At 9 weeks following ACLT, collagen orientation was significantly (p < 0.05) altered and proteoglycan content was significantly (p < 0.05) reduced in the superficial zone cartilage matrix. These structural changes either in the extracellular or pericellular matrix (ECM and PCM) were also correlated significantly (p < 0.05) with chondrocyte width and height changes, thereby suggesting that chondrocyte deformation response to mechanical compression in early OA changes primarily because of alterations in matrix structure. However, compared to the normal group, proteoglycan content in the PCM from the ACLT group decreased less than that in the surrounding ECM. Therefore, PCM could play a key role to protect excessive chondrocyte deformations in the ACLT group. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:342-350, 2018.

Three dimensional mesoscale analysis of translamellar cross-bridge morphologies in the annulus fibrosus using optical coherence tomography.

The defining characteristic of the annulus fibrosus (AF) of the intervertebral disc (IVD) has long been the lamellar structures that consist of highly ordered collagen fibers arranged in alternating oblique angles from one layer to the next. However, a series of recent histologic studies have demonstrated that AF lamellae contain elastin- and type VI collagen-rich secondary "cross-bridge" structures across lamellae. In this study, we use optical coherence tomography (OCT) to elucidate the three-dimensional (3-D) morphologies of these translamellar cross-bridges in AF tissues. Mesoscale volumetric images by OCT revealed a 3-D network of heterogeneously distributed cross-bridges. The results of this study confirm the translamellar cross-bridge is identifiable as a distinguishable structure, which lies in the interbundle space of adjacent lamellae and crisscrosses multiple lamellae in the radial direction. In contrast to previously proposed models extrapolated from 2-D sections, results from this current study show that translamellar cross-bridges exist as a complex, interconnected network. We also found much greater variation in lengths of cross-bridges within the interbundle space of lamellae (0.8-1.4 mm from the current study versus 0.3-0.6 mm from 2-D sections). OCT-based 3-D morphology of translamellar cross-bridge provides novel insight into the AF structure.

The effect of compressive loading magnitude on in situ chondrocyte calcium signaling.

Chondrocyte metabolism is stimulated by deformation and is associated with structural changes in the cartilage extracellular matrix (ECM), suggesting that these cells are involved in maintaining tissue health and integrity. Calcium signaling is an initial step in chondrocyte mechanotransduction that has been linked to many cellular processes. Previous studies using isolated chondrocytes proposed loading magnitude as an important factor regulating this response. However, calcium signaling in the intact cartilage differs compared to isolated cells. The purpose of this study was to investigate the effect of loading magnitude on chondrocyte calcium signaling in intact cartilage. We hypothesized that the percentage of cells exhibiting at least one calcium signal increases with increasing load. Fully intact rabbit femoral condyle and patellar bone/cartilage samples were incubated in calcium-sensitive dyes and imaged continuously under compressive loads of 10-40 % strain. Calcium signaling was primarily associated with the dynamic loading phase and greatly increased beyond a threshold deformation of about 10 % nominal tissue strain. There was a trend toward more cells exhibiting calcium signaling as loading magnitude increased (p = 0.133). These results provide novel information toward identifying mechanisms underlying calcium-dependent signaling pathways related to cartilage homeostasis and possibly the onset and progression of osteoarthritis.

A computational model to describe the regional interlamellar shear of the annulus fibrosus.

Interlamellar shear may play an important role in the homeostasis and degeneration of the intervertebral disk. Accurately modeling the shear behavior of the interlamellar compartment would enhance the study of its mechanobiology. In this study, physical experiments were utilized to describe interlamellar shear and define a constitutive model, which was implemented into a finite element analysis. Ovine annulus fibrosus (AF) specimens from three locations within the intervertebral disk (lateral, outer anterior, and inner anterior) were subjected to in vitro mechanical shear testing. The local shear stress-stretch relationship was described for the lamellae and across the interlamellar layer of the AF. A hyperelastic constitutive model was defined for interlamellar and lamellar materials at each location tested. The constitutive models were incorporated into a finite element model of a block of AF, which modeled the interlamellar and lamellar layers using a continuum description. The global shear behavior of the AF was compared between the finite element model and physical experiments. The shear moduli at the initial and final regions of the stress-strain curve were greater within the lamellae than across the interlamellar layer. The difference between interlamellar and lamellar shear was greater at the outer anterior AF than at the inner anterior region. The finite element model was shown to accurately predict the global shear behavior or the AF. Future studies incorporating finite element analysis of the interlamellar compartment may be useful for predicting its physiological mechanical behavior to inform the study of its mechanobiology.

Extracellular matrix integrity affects the mechanical behaviour of in-situ chondrocytes under compression.

Cartilage lesions change the microenvironment of cells and may accelerate cartilage degradation through catabolic responses from chondrocytes. In this study, we investigated the effects of structural integrity of the extracellular matrix (ECM) on chondrocytes by comparing the mechanics of cells surrounded by an intact ECM with cells close to a cartilage lesion using experimental and numerical methods. Experimentally, 15% nominal compression was applied to bovine cartilage tissues using a light-transmissible compression system. Target cells in the intact ECM and near lesions were imaged by dual-photon microscopy. Changes in cell morphology (N(cell)=32 for both ECM conditions) were quantified. A two-scale (tissue level and cell level) Finite Element (FE) model was also developed. A 15% nominal compression was applied to a non-linear, biphasic tissue model with the corresponding cell level models studied at different radial locations from the centre of the sample in the transient phase and at steady state. We studied the Green-Lagrange strains in the tissue and cells. Experimental and theoretical results indicated that cells near lesions deform less axially than chondrocytes in the intact ECM at steady state. However, cells near lesions experienced large tensile strains in the principal height direction, which are likely associated with non-uniform tissue radial bulging. Previous experiments showed that tensile strains of high magnitude cause an up-regulation of digestive enzyme gene expressions. Therefore, we propose that cartilage degradation near tissue lesions may be due to the large tensile strains in the principal height direction applied to cells, thus leading to an up-regulation of catabolic factors.

Superficial collagen fibril modulus and pericellular fixed charge density modulate chondrocyte volumetric behaviour in early osteoarthritis.

The aim of this study was to investigate if the experimentally detected altered chondrocyte volumetric behavior in early osteoarthritis can be explained by changes in the extracellular and pericellular matrix properties of cartilage. Based on our own experimental tests and the literature, the structural and mechanical parameters for normal and osteoarthritic cartilage were implemented into a multiscale fibril-reinforced poroelastic swelling model. Model simulations were compared with experimentally observed cell volume changes in mechanically loaded cartilage, obtained from anterior cruciate ligament transected rabbit knees. We found that the cell volume increased by 7% in the osteoarthritic cartilage model following mechanical loading of the tissue. In contrast, the cell volume decreased by 4% in normal cartilage model. These findings were consistent with the experimental results. Increased local transversal tissue strain due to the reduced collagen fibril stiffness accompanied with the reduced fixed charge density of the pericellular matrix could increase the cell volume up to 12%. These findings suggest that the increase in the cell volume in mechanically loaded osteoarthritic cartilage is primarily explained by the reduction in the pericellular fixed charge density, while the superficial collagen fibril stiffness is suggested to contribute secondarily to the cell volume behavior.

Chondrocyte deformation under extreme tissue strain in two regions of the rabbit knee joint.

Articular cartilage and its native cells-chondrocytes-are exposed to a wide range of mechanical loading. Chondrocytes are responsible for maintaining the cartilage matrix, yet relatively little is known regarding their behavior under a complete range of mechanical loads or how cell mechanics are affected by region within the joint. The purpose of this study was to investigate chondrocyte deformations in situ under tissue loads ranging from physiological to extreme (0-80% nominal strain) in two regions of the rabbit knee joint (femoral condyles and patellae). Local matrix strains and cell compressive strains increased with increasing loads. At low loads the extracellular matrix (ECM) strains in the superficial zone were greater than the applied tissue strains, while at extreme loads, the local ECM strains were smaller than the applied strains. Cell compressive strains were always smaller than the applied tissue strains and, in our intact, in situ preparation, were substantially smaller than those previously found in hemi-cylindrical explants. This resulted in markedly different steady-state cell volume changes in the current study compared to those working with cartilage explants. Additionally, cells from different regions in the knee exhibited striking differences in deformation behavior under load. The current results suggest: (i) that the local extracellular and pericellular matrix environment is intimately linked to chondrocyte mechanobiology, protecting chondrocytes from potentially damaging strains at high tissue loads; and (ii) that cell mechanics are a function of applied load and local cartilage tissue structure.

In situ chondrocyte viscoelasticity.

It has been proposed, based on theoretical considerations, that the strain rate-dependent viscoelastic response of cartilage reduces local tissue and cell deformations during cyclic compressions. However, experimental studies have not addressed the in situ viscoelastic response of chondrocytes under static and dynamic loading conditions. In particular, results obtained from experimental studies using isolated chondrocytes embedded in gel constructs cannot be used to predict the intrinsic viscoelastic responses of chondrocytes in situ or in vivo. Therefore, the purpose of this study was to investigate the viscoelastic response of chondrocytes in their native environment under static and cyclic mechanical compression using a novel in situ experimental approach. Cartilage matrix and chondrocyte recovery in situ following mechanical compressions was highly viscoelastic. The observed in situ behavior was consistent with a previous study on in vivo chondrocyte mechanics which showed that it took 5-7 min for chondrocytes to recover shape and volume following virtually instantaneous cell deformations during muscular loading of the knee in live mice. We conclude from these results that the viscoelastic properties of cartilage minimize chondrocyte deformations during cyclic dynamic loading as occurs, for example, in the lower limb joints during locomotion, thereby allowing the cells to reach mechanical and metabolic homeostasis even under highly dynamic loading conditions.

Mechanically induced calcium signaling in chondrocytes in situ.

Changes in intracellular calcium (Ca(2+)) concentration, also known as Ca(2+) signaling, have been widely studied in articular cartilage chondrocytes to investigate pathways of mechanotransduction. Various physical stimuli can generate an influx of Ca(2+) into the cell, which in turn is thought to trigger a range of metabolic and signaling processes. In contrast to most studies, the approach used in this study allows for continuous real time recording of calcium signals in chondrocytes in their native environment. Therefore, interactions of cells with the extracellular matrix (ECM) are fully accounted for. Calcium signaling was quantified for dynamic loading conditions and at different temperatures. Peak magnitudes of calcium signals were greater and of shorter duration at 37°C than at 21°C. Furthermore, Ca(2+) signals were involved in a greater percentage of cells in the dynamic compared to the relaxation phases of loading. In contrast to the time-delayed signaling observed in isolated chondrocytes seeded in agarose gel, Ca(2+) signaling in situ is virtually instantaneous in response to dynamic loading. These differences between in situ and in vitro cell signaling responses might provide crucial insight into the role of the ECM in providing pathways of mechanotransduction in the intact cartilage that are absent in isolated cells seeded in gel constructs.

A depth-dependent model of the pericellular microenvironment of chondrocytes in articular cartilage.

Experimental studies suggest that the magnitude of chondrocyte deformation is much smaller than expected based on the material properties of extracellular matrix (ECM) and cells, and that this result could be explained by a structural unit, the chondron, that is thought to protect chondrocytes from large deformations in situ. We extended an existing numerical model of chondrocyte, ECM and pericellular matrix (PCM) to include depth-dependent structural information. Our results suggest that superficial zone chondrocytes, which lack a pericellular capsule (PC), are relatively stiff, and therefore are protected from excessive deformations, whereas middle and deep zone chondrocytes are softer but are protected by the PC that limits cell deformations in these regions. We conclude that cell deformations sensitively depend on the immediate structural environment of the PCM in a depth-dependent manner, and that the functional stiffness of chondrocytes in situ is much larger than experiments on isolated cells would suggest.

Osmotic loading of in situ chondrocytes in their native environment.

Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.

Mechanical loading of in situ chondrocytes in lapine retropatellar cartilage after anterior cruciate ligament transection.

The aims of this study were (i) to quantify chondrocyte mechanics in fully intact articular cartilage attached to its native bone and (ii) to compare the chondrocyte mechanics for cells in healthy and early osteoarthritis (OA) tissue. We hypothesized that cells in the healthy tissue would deform less for given articular surface pressures than cells in the early OA tissue because of a loss of matrix integrity in early OA and the associated loss of structural integrity that is thought to protect chondrocytes. Chondrocyte dynamics were quantified by measuring the deformation response of the cells to controlled loading of fully intact cartilage using a custom-designed confocal indentation system. Early OA was achieved nine weeks following transection of the anterior cruciate ligament (ACL) in rabbit knees. Experiments were performed on the retropatellar cartilage of early OA rabbit knees (four joints and 48 cells), the corresponding intact contralateral control knees (four joints and 48 cells) and knees from normal control rabbits (four joints and 48 cells). Nine weeks following ACL transection, articular cartilage of the experimental joints showed substantial increases in thickness, and progression towards OA as assessed using histological grading. Local matrix strains in the superficial zone were greater for the experimental (38 +/- 4%) compared with the contralateral (27 +/- 5%) and normal (28 +/- 4%) joints (p = 0.04). Chondrocyte deformations in the axial and depth directions were similar during indentation loading for all experimental groups. However, cell width increased more for the experimental cartilage chondrocytes (12 +/- 1%) than the contralateral (6 +/- 1%) and normal control chondrocytes (6 +/- 1%; p < 0.001). On average, chondrocyte volume increased with indentation loading in the early OA cartilage (8 +/- 3%, p = 0.001), while it decreased for the two control groups (-8 +/- 2%, p = 0.002 for contralateral and -8 +/- 1%, p = 0.004 for normal controls). We conclude from these results that our hypothesis of cell deformations in the early OA tissue was only partially supported: specifically, changes in chondrocyte mechanics in early OA were direction-specific with the primary axial deformations remaining unaffected despite vastly increased average axial matrix deformations. Surprisingly, chondrocyte deformations increased in early OA in specific transverse directions which have received little attention to date but might be crucial to chondrocyte signalling in early OA.

Osmotic loading of articular cartilage modulates cell deformations along primary collagen fibril directions.

Osmotic loading is known to modulate chondrocyte (cell) height, width and volume in articular cartilage. It is not known how cartilage architecture, especially the collagen fibril orientation, affects cell shape changes as a result of an osmotic challenge. Intact patellae of New Zealand white rabbits (n=6) were prepared for fluorescence imaging. Patellae were exposed to a hypotonic osmotic shock and cells were imaged before loading and 5-60 min after the osmotic challenge. Cell volumes and aspect ratios (height/width) were analyzed. A fibril-reinforced poroelastic swelling model with realistic primary collagen fibril orientations, i.e. horizontal, random and vertical orientation in the superficial, middle and deep zones, respectively and cells in different zones was used to estimate cell aspect ratios theoretically. As the medium osmolarity was reduced, cell aspect ratios decreased and volumes increased in the superficial zone of cartilage both experimentally (p<0.05) and theoretically. Theoretically determined aspect ratios of middle zone cells remained virtually constant, while they increased for deep zone cells as osmolarity was reduced. Findings of this study suggest that osmotic loading modulates chondrocyte shapes in accordance with the primary collagen fibril directions in articular cartilage.

Confocal microscopy indentation system for studying in situ chondrocyte mechanics.

Chondrocytes synthesize extracellular matrix molecules, thus they are essential for the development, adaptation and maintenance of articular cartilage. Furthermore, it is well accepted that the biosynthetic activity of chondrocytes is influenced by the mechanical environment. Therefore, their response to mechanical stimuli has been studied extensively. Much of the knowledge in this area of research has been derived from testing of isolated cells, cartilage explants, and fixed cartilage specimens: systems that differ in important aspects from chondrocytes embedded in articular cartilage and observed during loading conditions. In this study, current model systems have been improved by working with the intact cartilage in real time. An indentation system was designed on a confocal microscope that allows for simultaneous loading and observation of chondrocytes in their native environment. Cell mechanics were then measured under precisely controlled loading conditions. The indentation system is based on a light transmissible cylindrical glass indentor of 0.17 mm thickness and 1.64 mm diameter that is aligned along the focal axis of the microscope and allows for real time observation of live cells in their native environment. The system can be used to study cell deformation and biological responses, such as calcium sparks, while applying prescribed loads on the cartilage surface. It can also provide novel information on the relationship between cell loading and cartilage adaptive/degenerative processes in the intact tissue.