View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP) Universidade de São Paulo Biblioteca Digital da Produção Intelectual - BDPI Departamento de Física e Ciência Interdisciplinar - IFSC/FCI Artigos e Materiais de Revistas Científicas - IFSC/FCI 2010 The mobility of chondroitin sulfate in articular and artificial cartilage characterized by 13C magic-angle spinning NMR spectroscopy Biopolymers,Hoboken : John Wiley and Sons,v. 93, n. 6, p. 520-532, 2010 http://www.producao.usp.br/handle/BDPI/50068 Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo The Mobility of Chondroitin Sulfate in Articular and Artificial Cartilage TheCharacterized Mobility of Chondroitinby 13C Magic-Angle Sulfate in Spinning Articular NMR and Artificial Spectroscopy Cartilage Characterized by 13C Magic-Angle Spinning NMR Spectroscopy Holger A. Scheidt,1 Stephanie Schibur,1 Alvicler Magalha˜es,2 Eduardo R. de Azevedo,3 Tito J. Bonagamba,3 Ovidiu Pascui,4 Ronny Schulz,5 Detlef Reichert,4 Daniel Huster1 1 Institute of Medical Physics and Biophysics, University of Leipzig, Ha¨rtelstr. 16-18, D-04107 Leipzig, Germany 2 Instituto de Quı´mica, UNICAMP, Medical Department, Caixa Postal 6154, CEP 13084-971, Campinas, Sa˜o Paulo, Brazil 3 Instituto de Fı´sica de Sa˜o Carlos, Universidade Sa˜o Paulo, Caixa Postal 369, CEP 13560-970, Sa˜o Carlos, Sa˜o Paulo, Brazil 4 Department of Physics, Martin Luther University Halle-Wittenberg, D-06108 Halle, Germany 5 Department of Cell Techniques and Applied Stem Cell Biology, Center of Biotechnology and Biomedicine, University of Leipzig, D-04103 Leipzig, Germany Received 23 September 2009; revised 5 January 2010; accepted 5 January 2010 Published online 20 January 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21386 ABSTRACT: sulfate in natural and artificial cartilage are different. We have studied the molecular dynamics of one of the The nuclear relaxation times of chondroitin sulfate in major macromolecules in articular cartilage, chondroitin natural and tissue-engineered cartilage were modeled sulfate. Applying 13C high-resolution magic-angle using a broad distribution function for the motional spinning NMR techniques, the NMR signals of all rigid correlation times. Although the description of the macromolecules in cartilage can be suppressed, allowing microscopic molecular dynamics of the chondroitin the exclusive detection of the highly mobile chondroitin sulfate in natural and artificial cartilage required the sulfate. The technique is also used to detect the identical broad distribution functions for the correlation chondroitin sulfate in artificial tissue-engineered times of motion, significant differences in the correlation cartilage. The tissue-engineered material that is based on times of motion that are extracted from the model matrix producing chondrocytes cultured in a collagen gel indicate that the artificial tissue does not fully meet the should provide properties as close as possible to those of standards of the natural ideal. This could also be the natural cartilage. Nuclear relaxation times of the confirmed by macroscopic biomechanical elasticity chondroitin sulfate were determined for both tissues. measurements. Nevertheless, these results suggest that NMR is a useful tool for the investigation of the quality of Although T1 relaxation times are rather similar, the T2 # relaxation in tissue-engineered cartilage is significantly artificially engineered tissue. 2010 Wiley Periodicals, shorter. This suggests that the motions of chondroitin Inc. Biopolymers 93: 520–532, 2010. Keywords: 13C MAS NMR; relaxation rates; tissue engineering; molecular dynamics Correspondence to: Daniel Huster; e-mail: [email protected] Contract grant sponsor: DFG This article was originally published online as an accepted Contract grant number: HU 720/7-1 preprint. The ‘‘Published Online’’ date corresponds to the Contract grant sponsor: German Academic Exchange Service preprint version. You can request a copy of the preprint by Contract grant sponsor: FAPESP Contract grant number: JP-05/59571-0 emailing the Biopolymers editorial office at biopolymers@wiley. VC 2010 Wiley Periodicals, Inc. com 520 Biopolymers Volume 93 / Number 6 Mobility of Chondroitin Sulfate 521 INTRODUCTION rticular cartilage is a connective tissue that is essen- tial for the function of diarthrodial joints.1,2 It pro- vides an extremely low friction surface in freely moving joints. In addition, articular cartilage is also a material of remarkable elastic properties. It Acan sustain high pressures of up to 20 MPa that are created from the stress and strain of exercises as well as daily tasks such as stair climbing.3 From the physical point of view, car- tilage is a gel of several cross-linked biopolymers that con- tains a large fraction of water (70–80%).1,4,5 The major macromolecules in cartilage are proteins and polysaccha- rides. About 15–20% (by wet weight) of cartilage is collagen (mostly type II), which provides the basic three-dimensional architecture of the material and imposes tensile strength.4 The polysaccharides are mostly organized in proteoglycans that contain large amounts of glycosaminoglycans (mostly chondroitin sulfate and little keratan sulfate). Proteoglycans make up 7–10% of the wet weight of the tissue. These highly negatively charged macromolecules are responsible for the swelling of cartilage and its high osmotic pressure.6 Previ- ous work has shown that the molecules in cartilage are mo- bile on different timescales and with varying amplitudes.7–10 Altogether, these microscopic properties determine the vis- coelasticity of the tissue providing load bearing and shock absorbing properties.3 In addition to the macromolecules mentioned above, there are small leucine-rich repeat proteo- glycans like decorin and biglycan and also a number of other proteins that function as structural components and are essential for morphogenesis of the extracellular matrix (ECM) and the entire molecular architecture of cartilage.11,12 FIGURE 1 (A) Schematic representation of the molecular archi- All components of the ECM are synthesized by only one cell tecture in articular cartilage. (B) Chemical structures of linkage type, the chondrocytes, which account for about 1–5% of the of chondroitin-4-sulfate (left) and chondroitin-6-sulfate (right). cartilage volume. A schematical sketch of the molecular GlcUA denotes glucuronic acid and the nomenclature of the carbon architecture in articular cartilage is shown in Figure 1A. atoms is given for one carbon ring. (C) Schematic representation of The molecules of the ECM of cartilage are in a constant the chondroitin sulfate linkage to the core protein of the proteogly- process of decomposition and resynthesis. Nevertheless, car- can aggregates. tilage as an avascular tissue only has a very limited self-heal- ing capacity.13–15 In particular, larger cartilage defects that tissue engineering is not a trivial problem. In vitro,boththe occur due to trauma or due to continuous degeneration as a culture system and the growth conditions stimulate the carti- consequence of osteoarthritis or inflammatory diseases such as lage forming chondrocytes to change their expression pattern, rheumatoid arthritis have very limited chances to self-heal.16–18 phenotype, or biochemical properties.22 This represents a serious socioeconomic problem in countries A number of physical techniques have been applied to with high life expectancy.19 Tissue engineering of cartilage may study the molecular architecture of cartilage. Besides nuclear represent an interesting strategy to replace diseased or worn magnetic resonance (NMR) spectroscopy23,24 and magnetic cartilage.20,21 To this end, chondrocytes are seeded into poly- resonance imaging,24,25 for instance Fourier transformed meric scaffolds, cultured, and stimulated to produce the typical infrared spectroscopy (FTIR),26 atomic force microscopy cartilage ECM ex vivo.21 Obviously, the artificially grown tissue (AFM),27 and x-ray diffraction28,29 are popular methods for should provide the appropriate structure and properties of studying the biophysical properties of the tissue. These tech- native cartilage. However, three-dimensional ex vivo cartilage niques are also well suited to investigate the macromolecules Biopolymers 522 Scheidt et al. in artificial cartilage to provide parameters that would char- Based on literature on the dynamic properties of synthetic acterize the quality of the ex vivo-manufactured tissue. A polymers, it is plausible that internal molecular motions in careful assessment of the quality of a tissue-engineered carti- GAG sidechains may affect the bulk mechanical properties of lage graft before implantation represents a crucial step for cartilage. Even local motions (side-chain motions for exam- the success of the operation.15 Most studies on tissue engi- ple) may have a significant influence on the mechanical neering of cartilage rely on optical assays and immunohisto- response of synthetic and biopolymers.34–36 However, despite chemistry to detect the molecules of the ECM in artificial tis- the fact that there is no study that relates the dynamics of sue. Because of the extremely high sensitivity of these meth- GAGs to the mechanical properties of cartilage, there is strong ods, even traces of collagen and glycosaminoglycans can be evidence that the increase of aggrecan stiffness