Roman M. Natoli

Effects of multiple chondroitinase ABC applications on tissue engineered articular cartilage

Increasing tensile properties and collagen content is a recognized need in articular cartilage tissue engineering. This study tested the hypothesis that multiple applications of chondroitinase ABC (C‐ABC), a glycosaminoglycan (GAG) degrading enzyme, could increase construct tensile properties in a scaffold‐less approach for articular cartilage tissue engineering. Developing constructs were treated with C‐ABC at 2 weeks, 4 weeks, or both 2 and 4 weeks. At 4 and 6 weeks, construct sulfated GAG composition, collagen composition, and compressive and tensile biomechanical properties were assessed, along with immunohistochemistry (IHC) for collagens type I, II, and VI, and the proteoglycan decorin. At 6 weeks, the tensile modulus and ultimate tensile strength of the group treated at both 2 and 4 weeks were significantly increased over controls by 78% and 64%, reaching values of 3.4 and 1.4 MPa, respectively. Collagen concentration also increased 43%. Further, groups treated at either 2 weeks or 4 weeks alone also had increased tensile stiffness compared to controls. Surprisingly, though GAG was depleted in the treated groups, by 6 weeks there were no significant differences in compressive stiffness. IHC showed abundant collagen type II and VI in all groups, with no collagen type I. Further, decorin staining was reduced following C‐ABC treatment, but returned during subsequent culture. The results support the use of C‐ABC in cartilage tissue engineering for increasing tensile properties.

In situ mechanical properties of the chondrocyte cytoplasm and nucleus

The way in which the nucleus experiences mechanical forces has important implications for understanding mechanotransduction. Knowledge of nuclear material properties and, specifically, their relationship to the properties of the bulk cell can help determine if the nucleus directly experiences mechanical loads, or if it is a signal transduction mechanism secondary to cell membrane deformation that leads to altered gene expression. Prior work measuring nuclear material properties using micropipette aspiration suggests that the nucleus is substantially stiffer than the bulk cell [Guilak, F., Tedrow, J.R., Burgkart, R., 2000. Viscoelastic properties of the cell nucleus. Biochem. Biophys. Res. Commun. 269, 781-786], whereas recent work with unconfined compression of single chondrocytes showed a nearly one-to-one correlation between cellular and nuclear strains [Leipzig, N.D., Athanasiou, K.A., 2008. Static compression of single chondrocytes catabolically modifies single-cell gene expression. Biophys. J. 94, 2412-2422]. In this study, a linearly elastic finite element model of the cell with a nuclear inclusion was used to simulate the unconfined compression data. Cytoplasmic and nuclear stiffnesses were varied from 1 to 7 kPa for several combinations of cytoplasmic and nuclear Poissons ratios. It was found that the experimental data were best fit when the ratio of cytoplasmic to nuclear stiffness was 1.4, and both cytoplasm and nucleus were modeled as incompressible. The cytoplasmic to nuclear stiffness ratio is significantly lower than prior reports for isolated nuclei. These results suggest that the nucleus may behave mechanically different in situ than when isolated.

Traumatic loading of articular cartilage: Mechanical and biological responses and post-injury treatment

This review discusses a framework for studying injurious loading of articular cartilage, which can lead to post-traumatic osteoarthritis. The framework separates the mechanical from the biological response of the tissue to injury. The mechanical response is governed by the tissues biomechanical behavior and sets off mechano-transductive pathways. These pathways then determine the biological response. The mechanical response of cartilage to injury has been studied by analytical and computational models of injurious loading, joint contact, and surface fissuring. These models have identified shear and tensile stresses as important parameters governing articular cartilage failure in response to mechanical injury. Further, measurement of cartilages material properties during impact loading has shown that the tissue is significantly stiffer than predicted from quasi-static testing. In terms of the biological response, cell death and sulfated glycosaminoglycan (sGAG) loss from the tissue are early degradative changes that lead to decreased tissue function. These biological sequelae have also been the subject of targeted intervention strategies post-injury. Some success has been found for decreasing cell death and sGAG loss using various bioactive agents. The framework and treatments reviewed here may be useful starting points in the study of mechanical injury to other tissues.

Intracellular Na+ and Ca2+ modulation increases the tensile properties of developing engineered articular cartilage

OBJECTIVE Significant collagen content and tensile properties are difficult to achieve in tissue-engineered articular cartilage. The aim of this study was to investigate whether treating developing tissue-engineered cartilage constructs with modulators of intracellular Na(+) or Ca(2+) could increase collagen concentration and construct tensile properties. METHODS Inhibitors of Na(+) ion transporters and stimulators of intracellular Ca(2+) were investigated for their ability to affect articular cartilage development in a scaffoldless, 3-dimensional chondrocyte culture. Using a systematic approach, we applied ouabain (Na(+)/K(+)-ATPase inhibitor), bumetanide (Na(+)/K(+)/2Cl(-) tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca(2+) ionophore) to tissue-engineered constructs for 1 hour daily on days 10-14 of culture and examined the constructs at 2 weeks or 4 weeks. The gross morphology, biochemical content, and compressive and tensile mechanical properties of the constructs were assayed. RESULTS The results of these experiments showed that 20 microM ouabain, 0.3 microM ionomycin, or their combination increased the tensile modulus by 40-95% compared with untreated controls and resulted in an increased amount of collagen normalized to construct wet weight. In constructs exposed to ouabain, the increased percentage of collagen per construct wet weight was secondary to decreased glycosaminoglycan production on a per-cell basis. Treatment with 20 microM ouabain also increased the ultimate tensile strength of neo-tissue by 56-86% at 4 weeks. Other construct properties, such as construct growth and type I collagen production, were affected differently by Na(+) modulation with ouabain versus Ca(2+) modulation with ionomycin. CONCLUSION These data are the first to show that treatments known to alter intracellular ion concentrations are a viable method for increasing the mechanical properties of engineered articular cartilage and identifying potentially important relationships to hydrostatic pressure mechanotransduction. Ouabain and ionomycin may be useful pharmacologic agents for increasing tensile integrity and directing construct maturation.

Introduction to Continuum Biomechanics

Abstract This book is concerned with the study of continuum mechanics applied to biological systems, i.e., continuum biomechanics. This vast and exciting subject allows description of when a bone may fracture due to excessive loading, how blood behaves as both a solid and fluid, down to how cells respond to mechanical forces that lead to changes in their behavior, a process known as mechanotransduction. We have written for senior undergraduate students and first year graduate students in mechanical or biomedical engineering, but individuals working at biotechnology companies that deal in biomaterials or biomechanics should also find the information presented relevant and easily accessible.Table of Contents: Tensor Calculus / Kinematics of a Continuum / Stress / Elasticity / Fluids / Blood and Circulation / Viscoelasticity / Poroelasticity and Thermoelasticity / Biphasic Theory

Effects of doxycycline on articular cartilage GAG release and mechanical properties following impact.

The effects of doxycycline were examined on articular cartilage glycosaminoglycan (GAG) release and biphasic mechanical properties following two levels of impact loading at 1 and 2 weeks post‐injury. Further, treatment for two continuous weeks was compared to treatment for only the 1st week of a 2‐week culture period. Following impact at two levels, articular cartilage explants were cultured for 1 or 2 weeks with 0, 50, or 100 µM doxycycline. Histology, GAG release to the media, and creep indentation biomechanical properties were examined. The “High” (2.8 J) impact level had gross surface damage, whereas “Low” (1.1 J) impact was indiscernible from non‐impacted controls. GAG staining decreased after High impact, but doxycycline did not visibly affect staining. High impact resulted in decreased aggregate moduli at both 1 and 2 weeks and increased permeability at 2 weeks, but tissue mechanical properties were not affected by doxycycline treatment. At 1 week, High impact resulted in more GAG release compared to non‐impacted controls. However, following High impact, 100 µM doxycycline reduced cumulative GAG release at 1 and 2 weeks by 30% and 38%, respectively, compared to no treatment. Interestingly, there was no difference in GAG release comparing 2 weeks continuous treatment with 1 week on, 1 week off. These results support the hypothesis that doxycycline can mitigate GAG release from articular cartilage following impact loads. However, doxycycline was unable to prevent the loss of tissue stiffness observed post‐impact, presumably due to initial matrix damage resulting solely from mechanical trauma. Biotechnol. Bioeng. 2008;100: 506–515.

Ameliorating glycosaminoglycan (GAG) loss and cell death in articular cartilage following single-impact loading

Impact loading of articular cartilage leads to post-traumatic osteoarthritis (OA) through its effects on the cells and extracellular matrix (ECM) of the tissue. Studies have shown the level of impact or injurious compression correlates with increased cell death, degradation of the ECM, and detrimental changes in biomechanical properties [1]. Recently, several bioactive agents, such as P188 and IGF-I, have shown promising results by reducing cell death following injurious compression of cartilage explants [2, 3].Copyright