Biomechanical and histological changes secondary to aging in the human rotator cuff: A preliminary analysis.

The high failure rate of rotator cuff repair surgeries is positively correlated with age, yet the biomechanical changes to the tendons of the rotator cuff with age have not been described. As such, we sought to benchmark and characterize the biomechanical and histopathological properties with the accompanying gene expression of human rotator cuff tendons as a function of age and histopathological degeneration. All four rotator cuff tendons from fresh human cadaver shoulders underwent biomechanical, histopathological, and gene expression analyses. Following cadaver availability, samples were grouped into Younger (i.e., less than 36 years of age, n = 2 donors) and Aged (i.e., greater than 55 years of age, n = 3 donors) as a means of characterizing and quantifying the age-related changes exhibited by the tendons. Biomechanical testing and subsequent computational modeling techniques revealed both differences in properties between tendons and greater Young's moduli in the Younger tendons (supraspinatus 3.06x, infraspinatus 1.76x, subscapularis 1.25x, and teres minor 1.32x). Histopathological scoring using the semi-quantitative Bonar scoring scheme revealed a positive correlation with age across all tendons (r = 0.508, p < 0.001). These data contextualize the biomechanical and histopathological changes to tendons that occurs naturally with aging, highlighting the innate differences in biomechanical properties of all four rotator cuff tendons, as well as the difference in their degenerative trajectories. Additionally, the histopathological scoring revealed moderate signs of degeneration within the Younger supraspinatus tendons, suggesting tissue quality may decrease in this specific tendon in patients less than 40 years old, before clinical symptoms or tears.

Modified Alendronate Mitigates Mechanical Degradation of the Rotator Cuff in an Osteoporotic Ovine Model.

BACKGROUND: Osteoporosis is an independent risk factor for failure after arthroscopic rotator cuff repair. Since rerupture rates after rotator cuff repair are associated with decreased bone mineral density and bone microarchitecture, adaptations of biomechanical properties of the rotator cuff enthesis in patients with osteoporosis remain unclear. Additionally, the effects of osteogenic therapy carrier drugs used for the treatment of osteoporosis on rotator cuff structure and properties have not been previously documented. PURPOSE: To investigate the changes to soft tissue biomechanics and insertional structure secondary to osteoporosis with and without an osteogenic therapy carrier (ie, modified alendronate). STUDY DESIGN: Controlled laboratory study. METHODS: Biomechanical, histopathological, and microcomputed tomography analyses were performed on 20 shoulders obtained from 10 osteoporotic sheep randomly allocated to modified bisphosphonate (ie, alendronate) or control (ie, osteoporotic without treatment) groups; 6 shoulders from healthy sheep were utilized for comparison purposes. RESULTS: Tendons from the control group exhibited a 57% decrease in undeformed Young modulus as compared with the healthy group (P = .010). Tendons from the modified bisphosphonate treatment group exhibited a 229% increase in initial Young modulus as compared with the control group (P = .010). Marked changes within the tendon insertional organization were noted in both the control and the modified bisphosphonate treatment group samples as evidenced by increased interdigitation of the bone-mineralized fibrocartilaginous junction. The control samples exhibited a markedly paucicellular insertion, whereas the modified bisphosphonate treated tendons exhibited a hypercellular insertional region as compared with the healthy group. Both groups exhibited significantly (P < .01) decreased bone quality underlying the infraspinatus insertion, as evidenced by all microcomputed tomography outcome parameters. CONCLUSION: This work illuminates changes to rotator cuff tendon secondary to osteoporosis. Specifically, it revealed decreased tendon modulus and altered insertional structure in the osteoporotic samples. Secondarily, these data revealed increases in tendon modulus accompanied by increased cellularity within the tendon insertion region after systemic modified bisphosphonate injections. CLINICAL RELEVANCE: Bisphosphonate treatment may have a positive effect on the healing of the enthesis after rotator cuff repair.

Biomechanical, Morphological, and Biochemical Characteristics of Articular Cartilage of the Ovine Humeral Head.

OBJECTIVE: Shoulder pain is commonly attributed to rotator cuff injury or osteoarthritis. Ovine translational models are used to investigate novel treatments aimed at remedying these conditions to prevent articular cartilage degeneration and subsequent joint degradation. However, topographical properties of articular cartilage in the ovine shoulder are undefined. This study investigates the biomechanical, morphological, and biochemical attributes of healthy ovine humeral head articular cartilage and characterizes topographical variations between surface locations. DESIGN: Ten humeral heads were collected from healthy skeletally mature sheep and each was segregated into 4 quadrants using 16 regions of interest (ROIs) across the articular surface. Articular cartilage of each ROI was analyzed for creep indentation, thickness, and sulfated glycosaminoglycan (sGAG) and collagen quantity. Comparisons of each variable were made between quadrants and between ROIs within each quadrant. RESULTS: Percent creep, thickness, and sGAG content, but not collagen content, were significantly different between humeral head quadrants. Subregion analysis of the ROIs within each surface quadrant revealed differences in all measured variables within at least one quadrant. Percent creep was correlated with sGAG (r = -0.32, P = 0.0001). Collagen content was correlated with percent creep (r = 0.32, P = 0.0009), sGAG (r = -0.19, P = 0.049), and thickness (r = -0.19, P = 0.04). CONCLUSIONS: Topographical variations exist in mechanical, morphologic, and biochemical properties across the articular surface of the ovine humeral head. Recognizing this variability in ovine humeral head cartilage will provide researchers and clinicians with accurate information that could impact study outcomes.

Mechanical, biochemical, and morphological topography of ovine knee cartilage.

The knee is the most common site for translational cartilage research in sheep, though topographic features of articular cartilage across surfaces are unspecified. We aimed to characterize the mechanical, morphological, and biochemical properties of articular cartilage across ovine knee surfaces and document variations between and within surface locations. Regions of interest (ROIs) were delineated across surfaces of 10 healthy ovine knees. Articular cartilage at each ROI was measured for creep indentation, thickness, and glycosaminoglycan (GAG) and collagen content. Variables were compared between surface locations (trochlea, and lateral [LFC] and medial [MFC] femoral condyles) and between ROIs within each surface location. Correlations between variables were also assessed. Articular surface location had a significant effect on creep (P < .0001), thickness (P < .0001), and collagen (P = .0007), but not GAG (P = .28). Significant differences in percent creep between ROIs were found within the LFC (P < .0001), MFC (P < .0001), and trochlea (P = .0002). Cartilage thickness was different between ROIs within the LFC, MFC, and trochlea (all P < .0001). The LFC (P = .002) and trochlea (P = .01) each had significant differences in GAG between ROIs. Collagen content between ROIs was different within the LFC (P = .0003), MFC (P = .0005), and trochlea (P < .0001). Collagen content was correlated with thickness (r = -.55), percent creep (r = .47), and GAG (r = -.21). Percent creep was correlated with thickness (r = -.64) and GAG (r = -.19). Topographic variations in mechanical, morphological, and biochemical properties exist across knee cartilage surfaces in sheep. Recognition of this variability is important to optimize study protocols and improve accuracy of results.

Comparing Predictive Accuracy and Computational Costs for Viscoelastic Modeling of Spinal Cord Tissues.

The constitutive equation used to characterize and model spinal tissues can significantly influence the conclusions from experimental and computational studies. Therefore, researchers must make critical judgments regarding the balance of computational efficiency and predictive accuracy necessary for their purposes. The objective of this study is to quantitatively compare the fitting and prediction accuracy of linear viscoelastic (LV), quasi-linear viscoelastic (QLV), and (fully) nonlinear viscoelastic (NLV) modeling of spinal-cord-pia-arachnoid-construct (SCPC), isolated cord parenchyma, and isolated pia-arachnoid-complex (PAC) mechanics in order to better inform these judgements. Experimental data collected during dynamic cyclic testing of each tissue condition were used to fit each viscoelastic formulation. These fitted models were then used to predict independent experimental data from stress-relaxation testing. Relative fitting accuracy was found not to directly reflect relative predictive accuracy, emphasizing the need for material model validation through predictions of independent data. For the SCPC and isolated cord, the NLV formulation best predicted the mechanical response to arbitrary loading conditions, but required significantly greater computational run time. The mechanical response of the PAC under arbitrary loading conditions was best predicted by the QLV formulation.

Viscoelasticity of spinal cord and meningeal tissues.

Compared to the outer dura mater, the mechanical behavior of spinal pia and arachnoid meningeal layers has received very little attention in the literature. This is despite experimental evidence of their importance with respect to the overall spinal cord stiffness and recovery following compression. Accordingly, inclusion of the mechanical contribution of the pia and arachnoid maters would improve the predictive accuracy of finite element models of the spine, especially in the distribution of stresses and strain through the cord's cross-section. However, to-date, only linearly elastic moduli for what has been previously identified as spinal pia mater is available in the literature. This study is the first to quantitatively compare the viscoelastic behavior of isolated spinal pia-arachnoid-complex, neural tissue of the spinal cord parenchyma, and intact construct of the two. The results show that while it only makes up 5.5% of the overall cross-sectional area, the thin membranes of the innermost meninges significantly affect both the elastic and viscous response of the intact construct. Without the contribution of the pia and arachnoid maters, the spinal cord has very little inherent stiffness and experiences significant relaxation when strained. The ability of the fitted non-linear viscoelastic material models of each condition to predict independent data within experimental variability supports their implementation into future finite element computational studies of the spine. STATEMENT OF SIGNIFICANCE: The neural tissue of the spinal cord is surrounded by three fibrous layers called meninges which are important in the behavior of the overall spinal-cord-meningeal construct. While the mechanical properties of the outermost layer have been reported, the pia mater and arachnoid mater have received considerably less attention. This study is the first to directly compare the behavior of the isolated neural tissue of the cord, the isolated pia-arachnoid complex, and the construct of these individual components. The results show that, despite being very thin, the inner meninges significantly affect the elastic and time-dependent response of the spinal cord, which may have important implications for studies of spinal cord injury.

The development and validation of a numerical integration method for non-linear viscoelastic modeling.

Compelling evidence that many biological soft tissues display both strain- and time-dependent behavior has led to the development of fully non-linear viscoelastic modeling techniques to represent the tissue's mechanical response under dynamic conditions. Since the current stress state of a viscoelastic material is dependent on all previous loading events, numerical analyses are complicated by the requirement of computing and storing the stress at each step throughout the load history. This requirement quickly becomes computationally expensive, and in some cases intractable, for finite element models. Therefore, we have developed a strain-dependent numerical integration approach for capturing non-linear viscoelasticity that enables calculation of the current stress from a strain-dependent history state variable stored from the preceding time step only, which improves both fitting efficiency and computational tractability. This methodology was validated based on its ability to recover non-linear viscoelastic coefficients from simulated stress-relaxation (six strain levels) and dynamic cyclic (three frequencies) experimental stress-strain data. The model successfully fit each data set with average errors in recovered coefficients of 0.3% for stress-relaxation fits and 0.1% for cyclic. The results support the use of the presented methodology to develop linear or non-linear viscoelastic models from stress-relaxation or cyclic experimental data of biological soft tissues.

Comparison of in vivo and ex vivo viscoelastic behavior of the spinal cord.

Despite efforts to simulate the in vivo environment, post-mortem degradation and lack of blood perfusion complicate the use of ex vivo derived material models in computational studies of spinal cord injury. In order to quantify the mechanical changes that manifest ex vivo, the viscoelastic behavior of in vivo and ex vivo porcine spinal cord samples were compared. Stress-relaxation data from each condition were fit to a non-linear viscoelastic model using a novel characterization technique called the direct fit method. To validate the presented material models, the parameters obtained for each condition were used to predict the respective dynamic cyclic response. Both ex vivo and in vivo samples displayed non-linear viscoelastic behavior with a significant increase in relaxation with applied strain. However, at all three strain magnitudes compared, ex vivo samples experienced a higher stress and greater relaxation than in vivo samples. Significant differences between model parameters also showed distinct relaxation behaviors, especially in non-linear relaxation modulus components associated with the short-term response (0.1-1 s). The results of this study underscore the necessity of utilizing material models developed from in vivo experimental data for studies of spinal cord injury, where the time-dependent properties are critical. The ability of each material model to accurately predict the dynamic cyclic response validates the presented methodology and supports the use of the in vivo model in future high-resolution finite element modeling efforts. STATEMENT OF SIGNIFICANCE: Neural tissues (such as the brain and spinal cord) display time-dependent, or viscoelastic, mechanical behavior making it difficult to model how they respond to various loading conditions, including injury. Methods that aim to characterize the behavior of the spinal cord almost exclusively use ex vivo cadaveric or animal samples, despite evidence that time after death affects the behavior compared to that in a living animal (in vivo response). Therefore, this study directly compared the mechanical response of ex vivo and in vivo samples to quantify these differences for the first time. This will allow researchers to draw more accurate conclusions about spinal cord injuries based on ex vivo data (which are easier to obtain) and emphasizes the importance of future in vivo experimental animal work.

Implantable microelectromechanical sensors for diagnostic monitoring and post-surgical prediction of bone fracture healing.

The relationship between modern clinical diagnostic data, such as from radiographs or computed tomography, and the temporal biomechanical integrity of bone fracture healing has not been well-established. A diagnostic tool that could quantitatively describe the biomechanical stability of the fracture site in order to predict the course of healing would represent a paradigm shift in the way fracture healing is evaluated. This paper describes the development and evaluation of a wireless, biocompatible, implantable, microelectromechanical system (bioMEMS) sensor, and its implementation in a large animal (ovine) model, that utilized both normal and delayed healing variants. The in vivo data indicated that the bioMEMS sensor was capable of detecting statistically significant differences (p-value <0.04) between the two fracture healing groups as early as 21 days post-fracture. In addition, post-sacrifice micro-computed tomography, and histology data demonstrated that the two model variants represented significantly different fracture healing outcomes, providing explicit supporting evidence that the sensor has the ability to predict differential healing cascades. These data verify that the bioMEMS sensor can be used as a diagnostic tool for detecting the in vivo course of fracture healing in the acute post-treatment period.

Nonlinear viscoelastic characterization of the porcine spinal cord.

Although quasi-static and quasi-linear viscoelastic properties of the spinal cord have been reported previously, there are no published studies that have investigated the fully (strain-dependent) nonlinear viscoelastic properties of the spinal cord. In this study, stress relaxation experiments and dynamic cycling were performed on six fresh porcine lumbar cord specimens to examine their viscoelastic mechanical properties. The stress relaxation data were fitted to a modified superposition formulation and a novel finite ramp time correction technique was applied. The parameters obtained from this fitting methodology were used to predict the average dynamic cyclic viscoelastic behavior of the porcine cord. The data indicate that the porcine spinal cord exhibited fully nonlinear viscoelastic behavior. The average weighted root mean squared error for a Heaviside ramp fit was 2.8 kPa, which was significantly greater (p<0.001) than that of the nonlinear (comprehensive viscoelastic characterization method) fit (0.365 kPa). Further, the nonlinear mechanical parameters obtained were able to accurately predict the dynamic behavior, thus exemplifying the reliability of the obtained nonlinear parameters. These parameters will be important for future studies investigating various damage mechanisms of the spinal cord and studies developing high-resolution finite elements models of the spine.

Nonlinear viscoelasticty plays an essential role in the functional behavior of spinal ligaments.

Despite the significant role ligament viscoelasticity plays in functional spinal biomechanics, relatively few studies have been performed to develop constitutive models that explicitly characterize this complex behavior. Unfortunately, the application and interpretation of these previous models are limited due to the use of simplified (quasi-linear) viscoelastic formulations or characterization techniques that have been shown to affect the predictive accuracy of the fitted coefficients. In order to surmount these previous limitations, the current study presents the application of a novel fitting technique (applied to stress relaxation experiments) and nonlinear viscoelastic constitutive formulation to human cervical spine anterior longitudinal ligament (ALL), posterior longitudinal ligament (PLL) and ligamentum flavum (LF). The fitted coefficients were validated by quantifying the ability of the constitutive equation to predict an independent cyclic data set across multiple physiologic strain amplitudes and frequencies. The resulting validated constitutive formulation indicated that the strain-dependent viscoelastic behavior of the longitudinal ligaments (ALL and PLL) was dominated by both the short-term (t=0.1s) and the steady-state (as t→∞) behavior. Conversely, the LF exhibited consistent relaxation behavior across the investigated temporal spectrum. From these data, it can be hypothesized that the unique strain-dependent temporal behavior of these spinal ligaments may be a functional adaptation that minimizes muscular expenditure during quasi-static postures while maximizing structural stability of the spine during transient loading events.

Viscoelastic effects during loading play an integral role in soft tissue mechanics.

Viscoelastic relaxation during tensioning is an intrinsic protective mechanism of biological soft tissues. However, current viscoelastic characterization methodologies for these tissues either negate this important behavior or provide correction methods that are severely restricted to a specific viscoelastic formulation and/or assume an a priori (linear) strain ramp history. In order to address these shortcomings, we present a novel finite ramp time correction method for stress relaxation experiments (to incorporate relaxation manifested during loading) that is independent of a specific viscoelastic formulation and can accommodate an arbitrary strain ramp history. We demonstrate transferability of our correction method between viscoelastic formulations by applying it to quasi-linear viscoelastic (QLV) and fully nonlinear viscoelastic constitutive equations. The errors associated with currently accepted methodologies for QLV and fully nonlinear viscoelastic formulations are elucidated. Our correction method is validated by demonstrating the ability of its fitted parameters to predict an independent cyclic experiment across multiple strain amplitudes and frequencies. The results presented herein: (i) indicate that our correction method significantly reduces the errors associated with previous methodologies; and (ii) demonstrate the necessity for the use of a fully nonlinear viscoelastic formulation, which incorporates relaxation manifested during loading, to model the viscoelastic behavior of biological soft tissues.

Experimental characterization and finite element implementation of soft tissue nonlinear viscoelasticity.

Finite element (FE) models of articular joint structures do not typically implement the fully nonlinear viscoelastic behavior of the soft connective tissue components. Instead, contemporary whole joint FE models usually represent the transient soft tissue behavior with significantly simplified formulations that are computationally tractable. The resultant fidelity of these models is greatly compromised with respect to predictions under temporally varying static and dynamic loading regimes. In addition, models based upon experimentally derived nonlinear viscoelastic coefficients that do not account for the transient behavior during the loading event(s) may further reduce the model's predictive accuracy. The current study provides the derivation and validation of a novel, phenomenological nonlinear viscoelastic formulation (based on the single integral nonlinear superposition formulation) that can be directly inputted into FE algorithms. This formulation and an accompanying experimental characterization technique, which incorporates relaxation manifested during the loading period of stress relaxation experiments, is compared to a previously published characterization method and validated against an independent analytical model. The results demonstrated that the static and dynamic FE approximations are in good agreement with the analytical solution. Additionally, the predictive accuracy of these approximations was observed to be highly dependent upon the experimental characterization technique. It is expected that implementation of the novel, computationally tractable nonlinear viscoelastic formulation and associated experimental characterization technique presented in the current study will greatly improve the predictive accuracy of the individual connective tissue components for whole joint FE simulations subjected to static and dynamic loading regimes.

Mechanical comparison of two suture constructs for extra-capsular stifle stabilization.

OBJECTIVE: Mechanical evaluation of 2 suture constructs for extracapsular stifle stabilization. STUDY DESIGN: In vitro study. SAMPLE POPULATION: Crimped interlocking loop constructs (ILC) of 45 kg nylon leader line (NLL) and Orthofiber® (OF). METHODS: ILC were tightened to 100 N, then crimp secured. Ramp to failure (n=10/group)-Data were derived from force/displacement plots. Stress-relaxation testing (n=10/group)-ILC's were nondestructively loaded and held at resultant displacement as force data were recorded. Incremental, cyclic loading (n=10/group)-ILC's were loaded (5 cycles/set) starting at 100 N and incrementally increased by 50 N (1 and 3 Hz protocols). Loop tension and elongation were recorded after each set. RESULTS: Ramp to failure-initial loop tension was similar (NLL 75.5 ± 9.5 N; OF 68.7 ± 10.4 N, P=.140). Tested OF constructs were stiffer (NLL 125.7 ± 4.0; OF 234.6 ± 25.0 N/mm, P≤.001), had lower yield load (NLL 193.6 ± 13.8; OF 137.3 ± 94.3 N, P≤.001), lower peak load (NLL 873.7 ± 68.6; OF 653.6 ± 80.2 N, P≤.001), and lower elongation at failure (NLL 19.1 ± 1.4; OF 5.2 ± 1.0 mm, P≤.001) and at yield (NLL 1.52 ± 0.2; OF 0.3 ± 0.6 mm, P=.003) than NLL constructs. Yield in NLL ILC's was variable knot tightening/crimp slippage, but only crimp-suture slippage in OF. Stress-relaxation testing-OF demonstrated greater relaxation. Incremental, cyclic loading-induced ILC elongation and tension loss in both groups, independent of loading frequency. NLL lost tension at lower rate, but elongated more than OF. CONCLUSIONS: NLL construct is mechanically superior to OF construct.

Human cervical spine ligaments exhibit fully nonlinear viscoelastic behavior.

Spinal ligaments provide stability and contribute to spinal motion patterns. These hydrated tissues exhibit time-dependent behavior during both static and dynamic loading regimes. Therefore, accurate viscoelastic characterization of these ligaments is requisite for development of computational analogues that model and predict time-dependent spine behavior. The development of accurate viscoelastic models must be preceded by rigorous, empirical evidence of linear viscoelastic, quasi-linear viscoelastic (QLV) or fully nonlinear viscoelastic behavior. This study utilized multiple physiological loading rates (frequencies) and strain amplitudes via cyclic loading and stress relaxation experiments in order to determine the viscoelastic behavior of the human lower cervical spine anterior longitudinal ligament, the posterior longitudinal ligament and the ligamentum flavum. The results indicated that the cyclic material properties of these ligaments were dependent on both strain amplitude and frequency. This strain amplitude-dependent behavior cannot be described using a linear viscoelastic formulation. Stress relaxation experiments at multiple strain magnitudes indicated that the shape of the relaxation curve was strongly dependent on strain magnitude, suggesting that a QLV formulation cannot adequately describe the comprehensive viscoelastic response of these ligaments. Therefore, a fully nonlinear viscoelastic formulation is requisite to model these lower cervical spine ligaments during activities of daily living.

Neurochemistry and electroanalytical probes.

Electroanalytical techniques have been applied to monitoring chemical events including neurotransmitter release during rodent behaviour and the release of zeptomoles of molecules from single cells. Transgenic mice models have been developed and studied to identify specific cell types in vitro. Characterization and surface modification of electroanalytical probes has enhanced the selectivity and sensitivity of measurements.

Separating vesicle fusion and exocytosis in hypertonic conditions.

The existence of an osmotic gradient between the vesicular contents of chromaffin and mast cells and the extracellular fluid plays a key role in the exocytotic process. Altering this gradient by elevating the external buffer osmolarity allows for the inhibition of exocytosis from cells and isolation and identification of prespike current events upon stimulation using cyclic voltammetry. Subsequent replacement of the osmotic gradient leads to release of the contents from fused vesicles.

The association of vesicular contents and its effects on release.

The oxidation of catecholamines with a carbon-fiber electrode can be used to monitor exocytosis at the single cell level at a variety of different types of cells. These measurements allow release to be followed from individual vesicles and have revealed several unique aspects concerning the coupling between release and storage. The strong association of the vesicular components in chromaffin cells dictates the time course of extrusion of the vesicle contents. Furthermore, liberation of the Ca(2+) normally stored within the vesicles can promote exocytosis without an external Ca(2+) source.

Dopamine transport into a single cell in a picoliter vial.

The analysis of chemical events in small volumes requires careful manipulation of samples and sensitive detection methods. Here, we describe the measurement of the neurotransmitter dopamine in a picoliter vial with electrochemical techniques. The vials were fabricated from fused-silica capillaries that provided a transparent container suitable for the observation and manipulation of a biological cell, sample solutions, and electrodes. Evaporation of the sample was prevented with a mineral oil layer, allowing for experiments lasting for several minutes. The small volume of these vials (100-200 pL) allows rapid mixing of all of the solution reagents. Similarly, the small volume allows exhaustive electrolysis of the vial contents with a 3-microm radius, disk-shaped carbon fiber microelectrode within 60 s. Fast-scan cyclic voltammetry at carbon fiber microelectrodes was used to monitor the concentration of analyte in the vial without depleting its contents. The concentration of dopamine introduced by pneumatic injection remained stable when sampled by cyclic voltammetry, and no evidence for adsorption to the walls was observed. However, when the vial contained a single HEK-293 cell transfected to express the dopamine transporter, the dopamine concentration decreased with time at a rate consistent with the uptake kinetics mediated by the transporter located on the cell membrane.

Temporal separation of vesicle release from vesicle fusion during exocytosis.

During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation.