Alan J. Grodzinsky

Fluorometric assay of DNA in cartilage explants using Hoechst 33258

A simple two-step fluorometric assay of DNA in cartilage explants, utilizing the bisbenzimidazole dye Hoechst 33258, is described. Cartilage explants were prepared for assay by digestion with papain. Aliquots of the digest were mixed with dye solution, and the fluorescence emission measured. The enhancement in fluorescence of dye was specific for DNA, as demonstrated by 97% sensitivity to DNase and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in fluorescence independent of the presence of papain-digested cartilage. By performing the assay on isolated chondrocytes, the cellular content of DNA was computed to be 7.7 pg per chondrocyte. The assay was stable for at least 2 h and sensitive to as little as 6 ng of DNA or equivalently less than 1000 cells. This procedure offers advantages over other established DNA assays of cartilage and may be especially useful in metabolic studies of cartilage explants.

Self-assembling peptide hydrogel fosters chondrocyte extracellular matrix production and cell division: Implications for cartilage tissue repair

Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix (ECM) accumulation in a three-dimensional geometry. A major challenge in choosing an appropriate scaffold for cartilage repair is the identification of a material that can simultaneously stimulate high rates of cell division and high rates of cell synthesis of phenotypically specific ECM macromolecules until repair evolves into steady-state tissue maintenance. We have devised a self-assembling peptide hydrogel scaffold for cartilage repair and developed a method to encapsulate chondrocytes within the peptide hydrogel. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time-dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically functional neo-tissue. Taken together, these results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like ECM within a three-dimensional cell culture for cartilage tissue repair.

Transcranial direct current stimulation: A computer-based human model study

OBJECTIVES Interest in transcranial direct current stimulation (tDCS) in clinical practice has been growing, however, the knowledge about its efficacy and mechanisms of action remains limited. This paper presents a realistic magnetic resonance imaging (MRI)-derived finite element model of currents applied to the human brain during tDCS. EXPERIMENTAL DESIGN Current density distributions were analyzed in a healthy human head model with varied electrode montages. For each configuration, we calculated the cortical current density distributions. Analogous studies were completed for three pathological models of cortical infarcts. PRINCIPAL OBSERVATIONS The current density magnitude maxima injected in the cortex by 1 mA tDCS ranged from 0.77 to 2.00 mA/cm(2). The pathological models revealed that cortical strokes, relative to the non-pathological solutions, can elevate current density maxima and alter their location. CONCLUSIONS These results may guide optimized tDCS for application in normal subjects and patients with focal brain lesions.

The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis

The healing of articular cartilage defects may be improved by the use of implantable three-dimensional matrices. The present study investigated the effects of four cross-linking methods on the compressive stiffness of collagen-glycosaminoglycan (CG) matrices and the interaction between adult canine articular chondrocytes and the matrix: dehydrothermal treatment (DHT), ultraviolet irradiation (UV), glutaraldehyde treatment (GTA), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The degree and kinetics of chondrocyte-mediated contraction, chondrocyte proliferation, and protein and glycosaminoglycan synthesis were evaluated over a four-week period in vitro. Cell-mediated contraction of the matrices varied with cross-linking: the most compliant DHT and UV matrices contracted the most (60% reduction in matrix diameter) and stiffest EDAC matrices contracted the least (30% reduction in matrix diameter). All cross-linking protocols permitted cell proliferation and matrix synthesis as measured by DNA content and radiolabeled sulfate and proline incorporation, respectively. During the first week in culture, a lower level of proliferation was seen in the GTA matrices but over the four-week culture period, the GTA and EDAC matrices provided for the greatest cell proliferation. On day 2, there was a significantly lower rate of 3H-proline incorporation in the GTA matrices (p<0.003) although at later time points, the EDAC and GTA matrices exhibited the highest levels of matrix synthesis. With regard to cartilage-specific matrix molecule synthesis, immunohistochemistry revealed a greater amount of type II collagen in DHT and UV matrices at the early time points. These findings serve as a foundation for future studies of tissue engineering of articular cartilage and the association of chondrocyte contraction and the processes of mitosis and biosynthesis.

The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression

The effects of streaming potential, fluid flow and hydrostatic pressure on chondrocyte biosynthesis were studied by comparing the spatial profiles of these physical stimuli to the profiles of biosynthesis within cartilage disks subjected to dynamic unconfined compression. The radial streaming potential was measured using compression frequencies and disk sizes relevant to studies of physical modulation of cartilage metabolism; a general analytical solution to the unconfined compression of a poroelastic cylinder with impermeable, rigid, adhesive platens was derived using potential theory. The solution with adhesive platen boundary conditions, using measured values of cartilage material properties, predicted streaming potentials that were much closer to experimental results between 0.001 and 1 Hz than a solution using frictionless platen boundary conditions. The predicted radial profiles of streaming potential gradient and fluid velocity (but not hydrostatic pressure) were similar to the previously reported radial dependence of proteoglycan synthesis induced by dynamic unconfined compression. Changes in stiffness associated with reduction of disk diameter suggested that the relative contributions of collagen and proteoglycans to cartilage mechanical properties may be a function of loading frequency in unconfined compression; such anisotropies may explain the remaining discrepencies between measured stiffness and stiffness predicted by the present model.

Cartilage electromechanics—II. A continuum model of cartilage electrokinetics and correlation with experiments

We have formulated a continuum model for linear electrokinetic transduction in cartilage. Expressions are derived for the streaming potential and streaming current induced by oscillatory, uniaxial confined compression of the tissue, as well as the mechanical stress generated by a current density or potential difference applied to the tissue. The experimentally observed streaming potential and current-generated stress response, measured on the same specimens, are compared with the predictions of the theory over a wide frequency range. The theory compares well with the data for reasonable values of cartilage intrinsic mechanical parameters and electrokinetic coupling coefficients. Experiments also show a linear relationship between the stimulus amplitude and the transduction response amplitude, within the range of stimulus amplitudes of interest. This observation is shown to be consistent with the predictions of the linear theory.

A molecular model of proteoglycan-associated electrostatic forces in cartilage mechanics

Measured values of the swelling pressure of charged proteoglycans (PG) in solution (Williams RPW, and Comper WD; Biophysical Chemistry 36:223, 1990) and the ionic strength dependence of the equilibrium modulus of PG-rich articular cartilage (Eisenberg SR, and Grodzinsky AJ; J Orthop Res 3: 148, 1985) are compared to the predictions of two models. Each model is a representation of electrostatic forces arising from charge present on spatially fixed macromolecules and spatially mobile micro-ions. The first is a macroscopic continuum model based on Donnan equilibrium that includes no molecular-level structure and assumes that the electrical potential is spatially invariant within the polyelectrolyte medium (i.e. zero electric field). The second model is based on a microstructural, molecular-level solution of the Poisson-Boltzmann (PB) equation within a unit cell containing a charged glycosaminoglycan (GAG) molecule and its surrounding atmosphere of mobile ions. This latter approach accounts for the space-varying electrical potential and electrical field between the GAG constituents of the PG. In computations involving no adjustable parameters, the PB-cell model agrees with the measured pressure of PG solutions to within experimental error (10%), whereas the ideal Donnan model overestimates the pressure by up to 3-fold. In computations involving one adjustable parameter for each model, the PB-cell model predicts the ionic strength dependence of the equilibrium modulus of articular cartilage. Near physiological ionic strength, the Donnan model overpredicts the modulus data by 2-fold, but the two models coincide for low ionic strengths (C0 < 0.025M) where the spatially invariant Donnan potential is a closer approximation to the PB potential distribution. The PB-cell model result indicates that electrostatic forces between adjacent GAGs predominate in determining the swelling pressure of PG in the concentration range found in articular cartilage (20-80 mg/ml). The PB-cell model is also consistent with data (Eisenberg and Grodzinsky, 1985, Lai WM, Hou JS, and Mow VC; J Biomech Eng 113: 245, 1991) showing that these electrostatic forces account for approximately 1/2 (290kPa) the equilibrium modulus of cartilage at physiological ionic strength while absolute swelling pressures may be as low as approximately 25-100kPa. This important property of electrostatic repulsion between GAGs that are highly charged but spaced a few Debye lengths apart allows cartilage to resist compression (high modulus) without generating excessive intratissue swelling pressures.

Static circumferential tangential modulus of human atherosclerotic tissue

The mechanical properties of atherosclerotic plaque may be of critical importance to the processes of plaque rupture, the most common antecedent of myocardial infarction. To investigate the effects of plaque structure and applied tensile stress on the static circumferential tangential modulus of atherosclerotic plaque, the stress-strain behavior of 26 human aortic intimal plaques was studied. Intimal plaques were collected during routine autopsies of 21 patients from the abdominal (n = 19) and thoracic (n = 2) aorta and were classified by histological analysis as cellular (n = 12), hypocellular (n = 9), and calcified (n = 5). At a physiologic applied circumferential tensile stress of 25 kPa, the tangential moduli of cellular, hypocellular, and calcified specimens were 927 +/- 468 kPa, 2312 +/- 2180 kPa, and 1466 +/- 1284 kPa, respectively. There was a nonsignificant difference in tangential modulus at 25 kPa stress between specimens classified as cellular and hypocellular (p = 0.098), cellular and calcified (p = 0.410), and hypocellular and calcified (p = 0.380). This is in marked contrast to the previously measured radial compressive behavior of plaque tissue, which showed that cellular, hypocellular, and calcified plaques were significantly different in their modulus. In tension, all 26 plaques tested demonstrated a statistically significant increase in tangential modulus with increasing applied circumferential stress. We conclude that the static circumferential tangential modulus of atherosclerotic plaque, unlike its radial compressive modulus, is not significantly affected by the degree of cellularity and calcification determined by histological characterization.(ABSTRACT TRUNCATED AT 250 WORDS)

Custom Design of the Cardiac Microenvironment With Biomaterials

Many strategies for repairing injured myocardium are under active investigation, with some early encouraging results. These strategies include cell therapies, despite little evidence of long-term survival of exogenous cells, and gene or protein therapies, often with incomplete control of locally-delivered dose of the factor. We propose that, ultimately, successful repair and regeneration strategies will require quantitative control of the myocardial microenvironment. This precision control can be engineered through designed biomaterials that provide quantitative adhesion, growth, or migration signals. Quantitative timed release of factors can be regulated by chemical design to direct cellular differentiation pathways such as angiogenesis and vascular maturation. Smart biomaterials respond to the local environment, such as protease activity or mechanical forces, with controlled release or activation. Most of these new biomaterials provide much greater flexibility for regenerating tissues ex vivo, but emerging technologies like self-assembling nanofibers can now establish intramyocardial cellular microenvironments by injection. This may allow percutaneous cardiac regeneration and repair approaches, or injectable-tissue engineering. Finally, materials can be made to multifunction by providing sequential signals with custom design of differential release kinetics for individual factors. Thus, new rationally-designed biomaterials no longer simply coexist with tissues, but can provide precision bioactive control of the microenvironment that may be required for cardiac regeneration and repair.

The effect of dynamic compression on the response of articular cartilage to insulin-like growth factor-I

Articular cartilage is routinely subjected to mechanical forces and to cell‐regulatory molecules. Previous studies have shown that mechanical stimuli can influence articular chondrocyte metabolic activity, and biochemical studies have shown that growth factors and cytokines control many of the same cell functions. Little is known, however, of the relationships or interplay, if any, between these two key components of the articular environment. This study investigated the comparative and interactive effects of low amplitude, sinusoidal, dynamic compression and insulin‐like growth factor‐I (IGF‐I), a polypeptide in synovial fluid that is anabolic for cartilage. In bovine patellofemoral cartilage explants, IGF‐I increased protein and proteoglycan synthesis 90% and 120%, respectively while dynamic compression increased protein and proteoglycan synthesis 40% and 90%, respectively. Stimulation by IGF‐I was significantly greater than by dynamic compression for both protein and proteoglycan synthesis. When applied together, the two stimuli enhanced protein and proteoglycan synthesis by 180% and 290%, respectively, a degree greater than that achieved by either stimulus alone. IGF‐I augmented protein synthesis with a time constant of 12.2 h. Dynamic compression increased protein synthesis with a time constant of 2.9 h, a rate significantly faster than that of IGF‐I, suggesting that these signals act via distinct cell activation pathways. When used together, dynamic compression and IGF‐I acted with a time constant of 5.6 h. Thus, dynamic compression accelerated the biosynthetic response to IGF‐I and increased transport of IGF‐I into the articular cartilage matrix, suggesting that, in addition to independently stimulating articular chondrocytes, cyclic compression may improve the access of soluble growth factors to these relatively isolated cells.