Dual proline labeling protocol for individual "baseline" and "response" biosynthesis measurements in human articular cartilage.

OBJECTIVE: The heterogeneity of biosynthesis in human-derived cartilage explants poses a challenge to its use in experiments. The aim of this study was to determine the consistency with which two consecutive measures of biosynthesis could be made in individual human articular cartilage explants using a dual proline radiolabeling protocol. METHODS: Full-thickness cartilage explants were harvested from young bovine or human (total knee replacement) tibial plateaus. Two consecutive measurements of biosynthesis were obtained by measuring (3)H-proline and (14)C-proline incorporation. Each sample's ratio of (14)C-/(3)H-proline incorporation was computed. For comparison to traditional experimental designs, the (14)C-proline incorporation ratio was computed for adjacent cartilage samples. The number of samples needed to observe a change in the proline incorporation ratio of 10, 20, and 50% was determined for both methods. RESULTS: The dual-label ratio was consistent across samples from the same plateau [95% confidence interval (CI): +/-20% (human) and +/-30% (bovine) of median]. Adjacent human sample pairs had much greater variability in their (14)C-proline incorporation (95% CI: +/-50% of median). Adjacent bovine sample pairs had CIs that were similar in magnitude to those for the dual-label approach. In the human plateaus, ratio changes of 10, 20 and 50% could be detected using dramatically fewer samples than the adjacent pair method. For bovine samples, the two methods required a similar number of samples per group. CONCLUSION: The consistency of the dual-label approach may overcome the difficulties in studying the effects of interventions on biosynthesis in human cartilage in vitro.

Author Correction: Computer keyboard interaction as an indicator of early Parkinson's disease.

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Computer keyboard interaction as an indicator of early Parkinson's disease.

Parkinson's disease (PD) is a slowly progressing neurodegenerative disease with early manifestation of motor signs. Objective measurements of motor signs are of vital importance for diagnosing, monitoring and developing disease modifying therapies, particularly for the early stages of the disease when putative neuroprotective treatments could stop neurodegeneration. Current medical practice has limited tools to routinely monitor PD motor signs with enough frequency and without undue burden for patients and the healthcare system. In this paper, we present data indicating that the routine interaction with computer keyboards can be used to detect motor signs in the early stages of PD. We explore a solution that measures the key hold times (the time required to press and release a key) during the normal use of a computer without any change in hardware and converts it to a PD motor index. This is achieved by the automatic discovery of patterns in the time series of key hold times using an ensemble regression algorithm. This new approach discriminated early PD groups from controls with an AUC = 0.81 (n = 42/43; mean age = 59.0/60.1; women = 43%/60%;PD/controls). The performance was comparable or better than two other quantitative motor performance tests used clinically: alternating finger tapping (AUC = 0.75) and single key tapping (AUC = 0.61).

Kinetics of the chondrocyte biosynthetic response to compressive load and release.

To gain insight regarding the rate at which cartilage tissue can sense and respond to a dynamic mechanical stimulus, we have examined the time-course of changes in biosynthetic activity following both the application and release of a static compressive stress. Cartilage harvested from the reserve zone of calf epiphyseal plate was subjected to unconfined static compressive stresses of 0, 0.25 and 0.5 MPa. Incorporation of [35S]sulfate and [3H]proline was measured during loading periods of less than 1 to 26 h and after preloading periods of 0.5, 2 or 12 h. During loading, total incorporation decreased to steady levels with time constants estimated to be 0.25-4 h (proline) and 1-5 h (sulfate). Proline incorporation exceeded control levels for 3 h after release of a 2 or 12 h preload. Sulfate incorporation remained depressed for at least 4 h after release of a 12 h preload and remained at control levels following release of 0.5 and 2 h preloads. We conclude that the modulation of proline incorporation by both loading and load release is faster than the modulation of sulfate incorporation. Furthermore, the response to unloading is not just the inverse of the response to loading; this nonlinearity suggests that the response to dynamic loading would not be determined simply by the time average component of the dynamic load.

Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts.

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its activity.

MRI techniques in early stages of cartilage disease.

Cartilage degenerative diseases affect millions of people. Our understanding of these diseases and our ability to establish efficacious treatment strategies have been confounded by the difficulty of nondestructively evaluating the state of cartilage. Imaging strategies that allow visualization of cartilage integrity would revolutionize the field by allowing us to visualize early stages of degeneration and thus to evaluate predisposing factors for cartilage disease and changes resulting from interventions (eg, therapies) in culture studies, tissue-engineered systems, animal models, and in vivo in humans. Here we briefly review current state-of-the-art MRI strategies relevant to understanding and following treatment in early cartilage degeneration. We review MRI as applied to the assessment of the whole joint, of cartilage as a whole (as an organ), of cartilage tissue, and of cartilage molecular composition and structure. Each of these levels is amenable to assessment by MRI and offers different information that, in the long run, will serve as an important element of cartilage imaging.

Magnetic resonance imaging of relative glycosaminoglycan distribution in patients with autologous chondrocyte transplants.

RATIONALE AND OBJECTIVES: Autologous chondrocyte transplantation (ACT) is a potential treatment for full-thickness chondral lesions in the knee. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) has recently been developed as a sensitive and specific measure of cartilage glycosaminoglycans (GAGs). Under the conditions of dGEMRIC, T1 is directly related to the GAG concentration. Our aim for this study was to demonstrate the potential of dGEMRIC to evaluate ACT implants. METHODS: Eleven ACT implants were studied 2 to 24 months postoperatively by dGEMRIC. T1 values from three regions of interest were obtained to examine GAG content (1) in the implant, (2) in native cartilage adjacent to the implant, and (3) in native cartilage further removed from the implant (as "control"). RESULTS: One implant failed and therefore was not included. Four of the implants were studied between 2 and 6 months postoperatively and showed low T1 (GAG), less than 80% of the control native cartilage. Five of the six implants studied between 12 and 24 months postoperativley showed T1 (GAG) comparable to (>80%) of control. One 18-month graft showed low T1 comparable to the surrounding native cartilage, with normal GAG seen in cartilage far from the graft site. The GAG index (T1 values of the graft normalized to control) from the group of implants 6 months or less was 59% +/- 5% of control, whereas those at 12 to 24 months were 91% +/- 18% of control. The two groups were statistically different with a P value of 0.005. CONCLUSIONS: The GAG level in grafts that were implanted for less than 12 months appeared to be lower than that in the remote cartilage. At 12 months or greater, the grafts in this study had GAG levels that were comparable to both the adjacent and remote cartilage. This preliminary study of ACT implants has shown that it is feasible to apply the dGEMRIC technique in patients with ACT as a way to obtain information related to the composition of grafts. These results provide motivation and the pilot data with which to design further clinical studies.

Dissociation of item and order memory following parietal cortex lesions in the rat.

Rats with parietal cortex lesions were tested for both item and order memory for a list of spatial events in a probe recognition procedure. Rats with parietal cortex lesions were impaired for all events within the item memory task but had good memory for the early events within the order memory task. These data suggest a dissociation of function between item and order memory for parietal cortex damaged animals. In conjunction with previous findings with rats with medial prefrontal cortex lesions, these data suggest that item and order memory can be coded and represented independently.

Dimensional growth and extracellular matrix accumulation by neonatal rat mandibular condyles in long-term culture.

Mandibular condyles in organ culture commonly have been used as a model system for examination of the factors that influence skeletal growth and development. The work reported here complements previously published histological studies by providing quantitative temporal information on growth and matrix accumulation. Condyles maintained for as long as 5 weeks in serum-free and 1% serum-supplemented culture media were found to remain viable and metabolically active as demonstrated by continued dimensional growth as well as cell and matrix accumulation. Growth occurred by a combination of cell proliferation, matrix synthesis and accumulation, and cell hypertrophy (with the latter two mechanisms dominating). Increases in tissue volume correlated directly with increased glycosaminoglycan content; both increased 7-fold over 5 weeks. In comparison with serum-free culture, after 35 days in medium containing 1% serum, glycosaminoglycan content was 24% lower and collagen content was 36% higher, whereas dry weight, condyle length, and DNA content were not significantly different; in addition, histological observation suggested that, for samples cultured with serum, chondrogenic phenotype had been lost from some regions. The temporal behavior for all growth parameters exhibited a transient phase 1-2 weeks in duration followed by a steady-state period in which dimensions and tissue constituents or content increased at a constant or near constant rate. Comparison of the rates of incorporation of [35S]sulfate with glycosaminoglycan content in serum-free cultures suggests that the loss of glycosaminoglycan occurs only initially or is negligible; therefore, under these baseline conditions, cartilage glycosaminoglycan content reflects the biosynthetic rate. The high degree of reproducibility seen during steady-state growth suggests that these data provide reliable baseline information and further supports the notion that this model system is useful for investigation of the effects of specific physical factors on in vitro growth and development.

Monitoring glycosaminoglycan replenishment in cartilage explants with gadolinium-enhanced magnetic resonance imaging.

We previously devised a magnetic resonance imaging method that allows for the nondestructive and quantitative determination of glycosaminoglycan concentration in excised cartilage. The technique measures the concentration of the charged contrast agent Gd-DTPA2- (gadolinium diethylenetriamine-pentaacetic acid) equilibrated within cartilage, from which the tissue distribution of glycosaminoglycan can be calculated. The goals of our study were to determine the practicality of nondestructively monitoring glycosaminoglycan concentration in cartilage explants over a long-term culture period and to determine if glycosaminoglycan could be restored to glycosaminoglycan-depleted cartilage explants maintained in long-term culture. To meet our objectives, we harvested bovine cartilage explants, treated them initially with trypsin to reduce the glycosaminoglycan concentration, and cultured them for as long as 8 weeks. Images depicting glycosaminoglycan concentration were calculated from magnetic resonance images acquired at selected intervals during the trypsinization process and the subsequent culture period. The results indicate that gadolinium-enhanced magnetic resonance imaging can follow the reduction of glycosaminoglycan concentration over the course of enzymatic digestion and the replenishment of glycosaminoglycan over several weeks of culture and that cultured cartilage explants are capable of restoring glycosaminoglycan to 85% of its initial concentration. Of particular interest, samples cultured for 5 weeks indicated a depth dependence of glycosaminoglycan regeneration to values similar to the initial physiologic distribution. Thus, this magnetic resonance imaging method may be a very powerful means for exploring the spatial and temporal evolution of glycosaminoglycan in cartilage.

Determination of fixed charge density in cartilage using nuclear magnetic resonance.

Many biomechanical and chemical properties of cartilage are dependent on the fixed charge density (FCD) of the extracellular matrix. In this study, nuclear magnetic resonance (NMR) spectroscopy was investigated as a nondestructive technique for determining FCD in cartilage. Sodium content was measured by NMR in cartilage explants and was compared with sodium content measured by inductively coupled plasma emission spectroscopy (ICP) in order to verify the total NMR visibility of sodium in cartilage. The ratio of NMR to ICP results was 1.02 +/- 0.04 (calf, mean +/- SD, n = 7) and 1.04 +/- 0.11 (adult bovine, n = 8). Sodium concentration as measured by NMR was then used with ideal Donnan theory to compute estimates of FCD. For calf articular cartilage (AC) near physiological conditions, calculated FCD was -0.28 +/- 0.03 M (n = 10). NMR measurements were then made for individual cartilage specimens sequentially equilibrated in baths of differing salt composition, pH, or ionic strength. For calf and adult AC, calculated FCD decreased dramatically between pH 3 and 2, with adult specimens becoming positively charged but calf tissue retaining a net negative charge. For calf AC equilibrated in 0.3-0.015 M NaCl, calculated FCD was observed to decrease slightly with decreasing bath ionic strength. For epiphyseal cartilage, FCD varied with the position of origin of the explant within the joint, ranging from -0.19 to -0.35 M in a manner that correlated with tissue glycosaminoglycan content. Preliminary NMR imaging experiments demonstrated similar variations of sodium concentration in intact ulnar epiphyseal cartilage. Collectively, these results demonstrate the ability of NMR to nondestructively follow FCD in cartilage. The technique is applicable to dynamic studies as well as to both in vitro and in vivo studies on living tissue.

Mechanical and physiochemical determinants of the chondrocyte biosynthetic response.

The relation between mechanical loading of cartilage and chondrocyte activity in vivo may be mediated by several physical transduction mechanisms including: cell deformation, hydrostatic pressure gradients, fluid flow, streaming currents, and physicochemical changes. We have developed an organ culture system designed to study chondrocyte biosynthetic response to such physical stimuli. This study focuses on the effects of static compression and physicochemical changes. Cartilage disks harvested from the reserve zone of the epiphyseal plate of 1-2-week-old calves were subjected to static compressive stresses of 0-3 MPa in unconfined compression and the incorporation of [35S]sulfate and [3H]proline was measured during the 12-h loading period. Incorporation of both proline and sulfate decreased monotonically with increasing stress. Compressive loading also produces physicochemical changes including a decreased water content and increased matrix fixed-charge density, with a concomitant increase in interstitial counterion concentrations (e.g., K+, H+) and decreased coion concentrations (e.g., SO4(2-). We therefore examined the possibility that specific changes in interstitial mobile ion concentrations may be linked to chondrocyte response to static compression by measuring biosynthesis in the absence of mechanical compression while independently altering the SO4(2-), K+, and H+ composition of the bathing medium. We found that proline and sulfate incorporation were strongly dependent on pH, but independent of [SO4(2-)] and [K+] in the range studied. These results suggest that compression-induced changes in local, interstitial pH may account for the observed biosynthetic response to static compression.

Osteoblast cytoskeletal modulation in response to mechanical strain in vitro.

The structural integrity of microfilaments has been shown to be necessary for the signal transduction of mechanical stimuli within osteoblasts. Qualitative and quantitative changes within the cytoskeleton of osteoblasts may therefore be crucial components of the signal transduction processes of these cells in response to mechanical stimulation. Avian osteoblasts were strained with a device that deforms a flexible, cell-laden membrane at a defined frequency and intensity in a uniform biaxial manner. We examined the effects of mechanical strain on the accumulation of protein and the expression of the major cytoskeletal elements and specific integrin-binding (arginine-glycine-aspartic acid) proteins of these cells. Mechanical strain increased the level of total extracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifiable effect on the accumulation of beta1 integrin subunit or collagen type I. An examination of the major elements of the cytoskeleton demonstrated that neither the level of actin nor that of the intermediate filament protein vimentin changed; however, the amount of tubulin decreased by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [35S]methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their altered synthesis. Messenger RNA analysis confirmed that the changes in accumulation and protein synthesis observed for vinculin, fibronectin, and osteopontin were controlled at a pretranslational level. Immunofluorescent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculin at the peripheral edges of the cells. In conclusion, the elevated rate of synthesis and the increased accumulation of vinculin and fibronectin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular matrix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.

Correlation between synthetic activity and glycosaminoglycan concentration in epiphyseal cartilage raises questions about the regulatory role of interstitial pH.

Current data provide compelling evidence that the pH of the interstitial fluid of cartilage is an important determinant of the metabolic activity of chondrocytes, and this has served as the basis for a mechanistic proposal whereby chondrocytes could sense mechanical compression. The objective of the current study was to test this hypothesis further by examining biosynthetic activity in cartilage as a function of glycosaminoglycan content, which is the major determinant of interstitial pH. On the basis of previous data, increased biosynthetic activity would be anticipated to correlate with a decreased glycosaminoglycan content and an elevated interstitial pH. In contrast to our expectations, we found that the biosynthetic activity (monitored by measurement of incorporation of sulfate and proline) was positively correlated with the glycosaminoglycan content of tissue. These results raise doubt as to whether interstitial pH provides a dominant mechanism for controlling the metabolism of chondrocytes.

Time-dependent changes in the response of cartilage to static compression suggest interstitial pH is not the only signaling mechanism.

The goal of the present study was to reexamine the role of interstitial pH in regulating the biosynthetic rate in cartilage tissue by addressing two research questions: (a) Do small, short-term changes in interstitial pH, induced independently by two different mechanisms (namely, by controlling the pH of the medium or by mechanical compression), result in biosynthetic rates commensurate with those expected from the "natural" relationship between interstitial pH and biosynthesis? and (b) Are the effects of changes in the pH of the medium or in compression the same for short-term (14-hour) and long-term (60-hour) exposures? Biosynthetic rates were estimated from incorporation of sulfate and proline into explants of bovine epiphyseal cartilage during the final 14 hours of culture. These rates decreased with decreasing pH of the medium, with increasing compression, and with decreasing native glycosaminoglycan content; or, expressed in terms of interstitial pH, acidification induced by compression or by lowering the pH of the medium resulted in a decreased biosynthetic rate, whereas interstitial acidification effected by increasing glycosaminoglycan content enhanced it. When the time for which tissue was exposed to changes in the pH of the medium was increased from 14 to 60 hours, the relationship between the biosynthetic rate and the pH remained constant whereas the relationship between the biosynthetic rate and compression was reversed. These data suggest that the transduction mechanisms underlying the response to pH of the medium and compression differ and that some adaptation or stimulation by modest levels of compression can occur with longer exposures. Interstitial pH is not the sole determinant of biosynthesis, and it cannot really account for the long-term response of cartilage tissue to static compression.

Diffusion of small solutes in cartilage as measured by nuclear magnetic resonance (NMR) spectroscopy and imaging.

The ability of water and solutes to move through the cartilage matrix is important to the normal function of cartilage and is presumed to be altered in degenerative diseases of cartilage such as osteoarthritis and rheumatoid arthritis. Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) techniques were used to measure a self diffusion coefficient (D) for small solutes in samples of explanted cartilage for diffusion times ranging from 13 ms to 2 s. With a diffusion time of 13 ms, the intratissue diffusivity of several small solutes (water, Na+, Li+, and CF3CO2-) was found consistently to be about 60% of the diffusivity of the same species in free solution. Equilibration of the samples at low pH (which titrates the charge groups so that the net matrix charge of -300 mM at pH 8 becomes approximately -50 mM at pH 2) did not affect the diffusivity of water or Na+. These data, and the similarity between the D in cartilage relative to free solution for water, anions, and cations, are consistent with the view that charge is not an important determinant of the intratissue diffusivity of small solutes in cartilage. With 35% compression, the diffusivity of water and Li+ dropped by 19 and 39%, respectively. In contrast, the diffusivity of water increased by 20% after treatment with trypsin (to remove the proteoglycans and noncollagenous proteins). These data and the lack of an effect of charge on diffusivity are consistent with D being dependent on the composition and density of the solid tissue matrix. A series of diffusion-weighted proton images demonstrated that D could be measured on a localized basis and that changes in D associated with an enzymatically depleted matrix could be clearly observed. Finally, evidence of restriction to diffusion within the tissue was found with studies in which D was measured as a function of diffusion time. The measured D for water in cartilage decreased with diffusion times ranging from 25 ms to 2 s, at which point the measured D was roughly 40% of the diffusivity in free solution. Although changes in matrix density by compression or digestion with trypsin led to a decrease or increase, respectively, in the measured D, the functional change in measured diffusivity with diffusion time remained essentially unchanged. In a different type of study, in which bulk transport could be observed over long periods of time, cartilage was submerged in 99% D2O and MRI studies were performed to demonstrate the bulk movement of water out of the cartilage matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Device for the application of a dynamic biaxially uniform and isotropic strain to a flexible cell culture membrane.

A large number of studies have demonstrated that mechanical perturbation modulates cellular metabolism; however, the systematic characterization of the molecular and cellular transduction mechanisms underlying mechanically induced metabolic modulation has been impeded, in part, by the limitations of the mechanical device. The objective of this investigation was to develop an in vitro experimental system that would provide independent control of the spatial and temporal biaxial strain distribution imposed on a flexible transparent tissue culture membrane that permits attachment, proliferation, and maintenance of the phenotypic expression of cultured embryonic osteoblasts. Such a device would permit a systematic investigation of the cellular response to specific, independently controlled parameters of mechanical deformation. Using a prototype device designed to impose a dynamic sinusoidal spatially isotropic biaxial strain profile, we confirmed experimentally that the strain was biaxially uniform and isotropic (radial = circumferential strain over the entire culture membrane) to within 14% (SD/mean) for the range of the peak strains tested (2.3-9.4%). Additionally, the uniformity was maintained at 1 Hz for at least 5 days of continuous operation. This experimental verification of the theoretically predicted isotropic strain profile suggests that the design principle is sound. Embryonic osteoblasts cultured on the flexible substrate proliferated and exhibited a temporal pattern of phenotypic expression (extracellular matrix accumulation and mineralization) comparable with that observed on polystyrene of tissue culture grade.

Toward an identification of mechanical parameters initiating periosteal remodeling: a combined experimental and analytic approach.

The ability of bone to adapt to its mechanical environment is well recognized, although the specific mechanical parameters initiating or maintaining the adaptive responses have yet to be identified. Recently introduced mathematical models offer the potential to aid in the identification of such parameters, although these models have not been well validated experimentally or clinically. We formulated a complementary experimental/analytic approach, using an animal model with a well-controlled mechanical environment combined with finite element modeling (FEM). We selected the functionally isolated turkey ulna, since the loading could be completely characterized and the periosteal adaptive responses subsequently monitored and quantified after four and eight weeks of loading. Known loads input into a three-dimensional, linearly elastic FEM of the ulna then permitted full-field mechanical characterization of the ulna. The FEM was validated against a normal strain-gaged turkey ulna, loaded in vivo in an identical fashion to the experimental ulnae. Twenty-four candidate mechanical parameters were then compared to the quantified adaptive responses, using statistical techniques. The data supported strain energy density, longitudinal shear stress, and tensile principal stress/strain as the mechanical parameters most likely related to the initiation of the remodeling response. Model predictions can now suggest new experiments, against which the predictions can be supported or falsified.

Quantitative measurement of the biological response of cartilage to mechanical deformation.

Cartilage functionality is defined, in part, in terms of the ability of the extracellular matrix to support a mechanical load. It has been shown that such mechanical loading can influence the biological response of the chondrocytes that are embedded in the extracellular matrix. Cultured tissue explants have served as useful models for studying such chondrocyte-mediated responses to mechanical deformation. The explant paradigm facilitates the control of mechanical and biological variables that may influence cellular behavior. This chapter presents the means for assessing the biologic response of cartilage to controlled mechanical stimuli, and lays the foundation to further explore the response of cartilage to mechanical stimuli in the presence of other factors, such as cytokines and growth factors (e.g., IL-1ß, TGF-ß, IGF).

Microsporidiosis in a young ostrich (Struthio camelus).

Microsporidia are obligate, intracellular, protozoan parasites of a wide variety of vertebrates and invertebrates. Confirmed reports of microsporidial infection of avian species are few (lovebirds, a parrot, and a group of budgerigar chicks). At slaughter, a 14-mo-old ostrich was found to have small intestinal serosal hemorrhages during postmortem inspection. Histologic examination of the small intestine revealed a chronic lymphoplasmacytic to purulent enteritis with mucosal hyperplasia, muscular hypertrophy, and numerous microsporidia that were located within the superficial enterocytes and the lamina propria. Microsporidia have a ubiquitous distribution in nature and are suspected as possible zoonotic agents.