Application of a tensile load directly to single Type I collagen monomers reduces the effective cleavage rate by specific collagenolytic enzymes (bacterial collagenase, cathepsin-K and matrix metalloproteinases) compared to unloaded controls. This is demonstrated by exposing multiple collagen molecules, in parallel, to degrading enzymes in a custom single molecule magnetic force assay.
Type I human collagen, covalently attached at either end to a superparamagnetic (SPM) bead and a glass slide surface, is loaded via a uniform magnetic field. The time to cleavage is estimated from probability distributions of collagen-tethered SPM bead ejection from the glass surface following addition of cleavage enzymes. The magnetic field strength is varied to alter the time to cleavage. The relationship between load and effective cleavage rate and the relationship between load and the time to complex formation (binding affinity) are determined.
Commercially available recombinant Type I human collagen (RhC1-003, Fibrogen, San Francisco, Calif.) is tethered between antibody functionalized superparamagnetic beads and a glass surface. For bead preparation, SPM beads (1.05±0.02 μm diameter, Invitrogen, Carlsbad, Calif.) are functionalized with antibodies to the C-terminus telopeptide of human collagen. The antibodies are attached to the beads via an EDAC linker using the PolyLink Protein Coupling Kit (Polysciences, Inc., Warrington, Pa.). LF-67 Human αI(1) carboxyl-telopeptide is added to EDAC linker coated beads in solution to allow antibody binding. The antibody functionalized beads are blocked with BSA and aggregation is minimized by exposure to Triton-X100.
The antibody functionalized beads are then exposed to collagen solution to promote binding. For glass preparation, 40 mm #1 borosilicate glass slides are plasma cleaned and silanized followed by direct addition of the linker GMBS (N-[g-maleimidobutyryloxy]succinimide ester)[GMBS, Pierce, Rockford, Ill.]. LF-116 Human α2(I) amino-telopeptide mixed with 10 ml 1% BSA/PBS solution is used to functionalize the glass. For antibody optimization, antibody dilutions of 1:100, 1:250, 1:500, 1:1000, 1:2000 and 1:5000 are tested and optimized, and optionally expanded into a binary search. These preliminary experiments are conducted by direct ELISA.
To apply mechanical load, the antibody-coated coverslip is placed into a microscope-mounted, closed-cell, flow chamber (FCS2, Bioptechs, Butler, Pa.) and exposed to the collagen-coated beads for incubation (1 hr at 25° C.). A stack of five neodymium magnets, which are calibrated for field strength vs. distance using Stoke's drag on a sphere, are positioned to produce a 10 pN force on the SPM beads. Collagen molecules reach their contour length of 300 nm at this level of applied load, and untethered beads are removed. The image plane of the microscope is set to the plane of the centerline of the tethered beads (nominally 800 nm above glass surface). Activated cleavage enzyme is introduced to the chamber at a rate and concentration that minimizes disturbance, yet provides rapid transport. Applied loads begin at a level that does not elongate a collagen monomer past its entropic regime (about 2 pN) and is extended into the elastic regime >10 pN. Load is increased depending on the detected sensitivity of the collagen/enzyme complex. To achieve forces greater than 10 pN, SPM beads of diameter greater than 1 μm are used. Controls are run with heat-inactivated cleavage enzyme. These experiments are repeated to include bacterial collagenase, MMP-1, MMP-8, MMP-13 (all Sigma, Mo.), and cathepsin-K (Biomol International Inc., PA). At least five different loads per enzyme concentration will be run and three different enzyme concentrations are run per load. Experiments are repeated three times.
Z position is determined using the method described in Part B, below.
A relationship is determined between cleavage rate and mechanical load/concentration for every enzyme that is tested.
Increasing tensile load on collagen monomers increases the time to cleavage.
Quantum dots (Invitrogen, Carlsbad, Calif.) are prepared according to the manufacturer's protocol, and bound to GMBS in the same fashion as antibody attachment as described in Part A, above. During a binding event, the MMP alters the length, z, of the collagen monomer or alters the Brownian excursion distance, δx. Collagen length change is measured as follows. Since the refractive index of the quantum dot and the experimental media are different, the point spread function (PSF) of the quantum dots varies based on depth in the media. By using a model of the predicted point spread function developed by Aquet et al. (Confocal, Multiphoton, and Nonlinear Microscope Imaging II (2005)), the z-position is determined with precision in the nanometer range by fitting the actual PSF to the model. A 60× objective is focused onto the collagen tethered SPM beads with the quantum dots glass-bound position out of focus. The quantum dot positions are determined with greater accuracy using defocused particles. Bead position relative to the focal plane is estimated by fitting a circle to the outline of the bead, and the PSF fit determines the position of the quantum dot. This distance is monitored for changes as enzyme is introduced into the chamber. Brownian motion changes are measured as follows. Since the catabolic enzymes bind tightly to the collagen molecule (stiffening) and comprise a significant percentage of collagen's molecular weight (33% for BC), binding alters the Brownian excursion. Thus, x-y position is monitored following the introduction of enzyme to the chamber.
A high-resolution camera (Retiga EXL, QI Imaging, 1394b, Mono, 14 bit cooled OC) is added to a Nikon TE2000E microscope, which enables vertical, spatial and temporal resolution to a level that allows detection of changes in the relative position of particles on the order of nanometers. Bead and quantum dot positions are reported as functions of time, and the relative displacement curves are examined for abrupt changes.
Strain applied to collagen monomers modifies the binding rate coefficient and the cleavage rate coefficient. Effective cleavage rates of collagen by enzymes are the result of a binding and cleavage event. Both of these events involve physical interaction of the enzyme with specific sites on the collagen monomer and are affected by the application of strain. The detection of the binding events allows a direct determination of the extent each event (binding or cleavage) is influenced by mechanical strain.
Atomistic-based multi-scale modeling is used for the chemomechanical behavior of tropocollagen monomers and the hierarchical structure of collagen fibrils, focusing on the effect of strain, enzymatic activity, and the coupling between strain and cleavage probability. The mechanical properties of tropocollagen molecules are investigated using steered molecular dynamics simulations using both a CHARMM force field as well as the new first principles based reactive force field ReaxFF. The ReaxFF model includes a first principles based description of all chemical bonds in the system (including breaking and formation of covalent bonds). This method captures chemomechanical coupling. The ReaxFF model bridges the scale from first principles quantum mechanical descriptions (limited to <200 atoms) to empirical CHARMM-like force fields and molecular mechanics models. In ReaxFF, the energy of atoms and molecules and their charges is not calculated based on simple distance-energy relations, but instead, is calculated dependent on the quantum mechanical state or bond orders.
The software THeBuScr (Triple-Helical collagen Building Script) is used to build a model of the tropocollagen molecule. This builder enables the generation of tropocollagen monomers with any desired sequence. The sequence is chosen based on those structures relevant to the cleavage domain in the collagen molecule. The MMP enzyme structures are taken from the protein data bank (PDB).
The tropocollagen molecule is subjected to traction along its principal axis using steered molecular dynamics (SMD). Other studies are focused on coupling between mechanical strain and the effect on the collagen network formation and stability. The interaction of a single tropocollagen molecule with MMP is studied under varying stain. The energy landscape of these atomic mechanisms is mapped and quantitative information is provided of how strain affects the interaction of the enzyme with the collagen. This series of computational experiments discerns that binding affinity and/or cleavage ability (modeled as the change in energy used to separate an alpha chain from the triple helix) is affected by the applied load. Using a combination with mesoscale simulations, entropic effects are captured that may play a key role at longer time-scales and, in the case of longer tropocollagen monomer sections, during assembly processes. The interaction and assembly of a large ensemble of >10,000 tropocollagen monomers is simulated under varying mechanical strain and under varying enzymatic activity.
Collagen fibrils are protected from enzymatic attack if they are “strained” appropriately. The methods described in Example 2 are modified by applying a strain field to the construct, creating a strain on the order of 1.5% to 4% where fibril retention is desired. Fibrils outside this range are preferentially degraded. Controllable strain is created by fixing the organized collagen gels to moveable grips or functionalized surfaces (see, e.g., Ruberti et al., Biochem. Biophys. Res. Commun. 336:483-489 (2005)). FIG. 25 shows two devices that are used to apply known loads to collagen constructs. Both devices allow high-powered optical observation and media access for enzyme and monomer cycling. These devices create uniaxial and tangential loads suitable for the production tendon-like and cornea-like