Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: towards the design of cartilage-specific functional contrast agents.

BACKGROUND: Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur. METHODS: Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone. RESULTS: Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde-dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes. CONCLUSIONS AND GENERAL SIGNIFICANCE: Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents.

Adsorption and distribution of fluorescent solutes near the articular surface of mechanically injured cartilage.

The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces.

Water binding energies of [Pb(amino acid-H)H2O]+ complexes determined by blackbody infrared radiative dissociation.

The water binding energies (E(0)) of eight deprotonated Pb(2+)-amino acid (Aa) complexes of the form [Pb(Aa-H)H(2)O](+) (Aa = Gly, Ala, Val, Leu, Ile, Phe, Glu, and Lys) were determined using blackbody infrared radiative dissociation (BIRD). A Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer was used to trap ions generated by electrospray ionization (ESI) in a "zero"-pressure (~10(-10) torr) cell where dissociation can only occur by absorption of thermally generated photons. Since the [Pb(Aa-H)H(2)O](+) complexes have relatively few vibrational degrees of freedom (36-78) and are within the slow-exchange kinetic limit, the master equation was solved to extract meaningful threshold dissociation energies and thermal unimolecular dissociation rate constants (k(uni)). The master equation analysis uses variable reaction coordinate transition state theory (VRC-TST) to minimize the Rice-Ramsperger-Kassel-Marcus (RRKM) dissociation rate constants. The determined water binding energies range from 76.6 to 113.6 kJ mol(-1), and agree well with 0 K dissociation energies calculated using the B3LYP/6-31+G(d,p) and MP2(full)/6-311++G(2d,2p)//B3LYP/6-31+G(d,p) methods. The relative strengths of the binding energies reflect the known structural isomers (A-, B-, C-, and D-type) of these [Pb(Aa-H)H(2)O](+) complexes.

Structures of bare and hydrated [Pb(aminoacid-H)]+ complexes using infrared multiple photon dissociation spectroscopy.

Infrared multiple-photon dissociation (IRMPD) spectroscopy was used to determine the gas-phase structures of deprotonated Pb(2+)/amino acid (Aa) complexes with and without a solvent molecule present. Five amino acid complexes with side chains containing only carbon and hydrogen (Ala, Val, Leu, Ile, Pro) and one with a basic side chain (Lys) were compared. These experiments demonstrated that all [Pb(Aa-H)](+) complexes have Pb(2+) covalently bound between the amine nitrogen and carbonyl oxygen. The nonhydrated complexes containing Ala, Val, Leu, Ile, and Pro are amine-deprotonated, whereas the one containing Lys is deprotonated at its carboxylic acid. The difference is attributed to the polar and basic side chain of lysine, which helps stabilize Pb(2+). IRMPD spectroscopy was also performed on the monohydrated analogues of the [Pb(Aa-H)](+) complexes. The [Pb(Aa-H)H(2)O](+) complexes, where Aa = Ala, Val, Leu, and Ile, exhibited two N-H stretches as well as a carboxylic acid O-H and a PbO-H stretch. Hence, their structures are monohydrated versions of the amine-deprotonated [Pb(Aa-H)](+) complexes where a proton transfer has occurred from the lead-bound water to the deprotonated amine. The IRMPD spectrum and calculations suggest that [Pb(Pro-H)H(2)O](+) has a hydrated carboxylate salt structure. The structure of [Pb(Lys-H)H(2)O](+) was also carboxyl-deprotonated, but Pb(2+) is bound to the carbonyl oxygen and the amine nitrogen, with one of the protons belonging to the water transferred to the basic side chain. This results in an intramolecular hydrogen bond that does not absorb in the region of the spectrum probed in these experiments. The IRMPD spectra and structural characterizations were confirmed and aided by infrared spectra calculated at the B3LYP/6-31+G(d,p) level of theory and 298 K enthalpies and Gibbs energies using the MP2(full)/6-311++G(2d,2p) method on the B3LYP geometries.