Measuring and testing – Testing of material
Reexamination Certificate
1999-08-11
2001-09-18
Raevis, Robert (Department: 2856)
Measuring and testing
Testing of material
Reexamination Certificate
active
06289753
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to assessing mechanical integrity of extracellular matrices such as cartilage and, more particularly, to a method for measuring the mechanical integrity of the collagen network in cartilage, and for modeling and assessing cartilage according to the measured collagen network mechanical properties.
BACKGROUND OF THE INVENTION
Since Ogston first proposed a model of connective tissue consisting of “a relatively coarse fibrous collagen network” that balances the osmotic pressure of a “molecular network of polysaccharide fibers trapped within it”, surprisingly little work has been done to use it to study the behavior of cartilage. (Ogston, A. G. (1970) in Chemistry and Molecular Biology of the Intracellular Matrix: The biological functions of the glycosaminoglycans Eds. (13alazs, E. A., Eds.), pp. 1231-1240, Academic Press, London).
Subsequent studies have been performed to measure the proteoglycan (PG) osmotic pressure, &pgr;
PG
, in normal and osteoarthritic (OA) cartilage specimens, yet, no comparable quantitative methods have been developed to characterize the integrity of the collagen network per se, such as its ability to “restrain” the PGs from swelling. See, Maroudas, A., Bayliss, M. T., and Venn, M. F. (1980)
Ann Rheum Dis
39, 514-23; Grushko, G., Schneiderman, R., and Maroudas, A. (1989)
Connective Tissue Research
19, 149-176; and Maroudas, A., Ziv, I., Weisman, N., and Venn, M. (1985)
Biorheology
22, 159-69, which are herein incorporated by reference. Consequently, no methods are available to characterize the PG and collagen network phases in situ.
Being able to characterize the PGs and the collagen network together, however, is particularly important in understanding the etiology of OA. Although there has been indirect evidence presented that suggests that the collagen network loses mechanical integrity early in OA whereas PG content and composition may not change appreciably, it has not possible to demonstrate this definitively since heretofore no methodology for characterizing both the state of the PGs and of the collagen network phases in situ in the same tissue specimen has been developed. See, Maroudas, A. and Venn, M. (1977)
Ann Rheum Dis
36, 399-406. Maroudas, A. (1976)
Nature
260, 808-9. Maroudas, A., Evans, H., and Almeida, L. (1973)
Ann. Rheum. Dis
. 32, 1-9.
More generally, experimental and theoretical tools to determine collagen network integrity are also needed to understand the functional consequences of a) endogenous structural changes in cartilage (e.g., that occur normally in normal and abnormal development, aging, degeneration, and disease), b) exogenous changes (e.g., following the addition of biochemical agents such as proteinases, or resulting from genetic manipulations), and c) inherent differences between cartilage tissues (e.g., between species; as well as between different joints, different locations on the same joint, and young and old individuals of the same species). In addition, there is a need to understand how environmental (chemical, mechanical, or electrical) stresses affect connective tissue structure and function. These stresses may be endogenous (e.g., occurring during locomotion), or exogenous (e.g., applied externally to tissue cultures in vitro or during a clinical evaluation).
Further, it is becoming increasingly important to evaluate the growth and viability of the collagen network within tissue-engineered connective tissues, both in vitro (e.g., in drug efficacy studies), and in vivo prior to and following their implantation. Without a quantitative measure of collagen network integrity together with the swelling characteristics of the PGs, the relationship between collagen network structure and tissue function cannot be established.
There is, therefore, a need for a quantitative methodology for determining the mechanical integrity of the collagen network in cartilage and of other extracellular matrices.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to measure the mechanical properties of a collagen network in cartilage.
Another object of the present invention is to model the mechanical properties of cartridge according to separate phases for the collagen network and for the proteoglycan constituents.
A further object is to assess the condition of cartilage according to the mechanical properties of the collagen network in the cartilage.
Yet a further object of the present invention is to assess the mechano-chemical state of both the proteoglycan and collagen network phases in cartilage.
Still another object is to identify cartilage pathology according to the mechanical properties of the collagen network in the cartilage.
Yet another object of the present invention is to measure the mechanical properties of a collagen network in cartilage according to an in vitro mechano-osmotic technique.
Still a further object is to measure the hydrostatic pressure caused by tensile stresses developed within the collagen network as a function of tissue hydration.
Another object is to provide measures of mechanical integrity of the collagen and proteoglycan phases from data acquired in the osmotic titration measurements.
The present invention achieves these and other objects, and overcomes limitations of the background art and the prior art, by providing a method for measuring the mechanical integrity of a collagen network in cartilage and of other extracellular matrices according to applying a known mechanical stress to the sample; measuring a quantity representing hydration of the sample; and providing the extracellular network recoil pressure according to the known applied mechanical stress and the independently determined proteoglycan osmotic pressure corresponding to the hydration.
In accordance with an embodiment of the present invention, an in vitro mechano-osmotic titration method for determining the collagen network tension (i.e., recoil pressure) for a given collagen network hydration is used. Collagen samples are equilibrated in a saline solution and weighed. Some of these samples are compressed with various known applied osmotic pressures values by dialyzing them against different polyethylene glycol (PEG) concentrations, and then are again weighed. Other samples are first swelled by immersing them in a hypotonic saline solution, then compressed using a PEG concentration sufficient to restore their original volume in physiological saline. Collagen network hydration is based on the measured volumes of the water, PG, and non-collagenous protein phases compared with the measured volume of collagen network phase. The proteoglycan osmotic pressure corresponding to the measured hydrations is independently determined according to proteoglycan osmotic pressure versus proteoglycan fixed charge density measurements, intra-fibrillar and extra-fibrillar water content determined from x-ray diffraction, and collagen content measurements. For each hydration, the collagen network recoil pressure is then calculated according to the independently determined proteoglycan osmotic pressure and to the known applied osmotic pressure. By determining the collagen network tension for a range of hydrations, the mechanical recoil pressure of the collagen network may be represented.
In accordance with a further embodiment of the present invention, the method may be used for diagnosing and/or monitoring the progression of diseases, such as osteoarthritis, preferably according to the collagen network stiffness, which for example, is shown to be reduced in osteoarthritic cartilage compared with normal cartilage. In accordance with a further embodiment of the present invention, the method may be used for assessing changes that occur normally in aging, which for example, is shown to increase collagen network stiffness. In accordance with yet a further embodiment of the present invention, the measured mechano-chemical properties of the collagen network in cartilage are incorporated into a mechanical model of cartilage in which the collagen network and proteoglycans are ma
Basser Peter J.
Maroudas Alice
Morgan & Finnegan L.L.P.
Raevis Robert
The United States of America as represented by the Department of
LandOfFree
Method for measuring mechanical properties of the collagen... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for measuring mechanical properties of the collagen..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for measuring mechanical properties of the collagen... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2549024