In Vitro Cartilage Wear Studies

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Over the past 15 years, studies aimed at exploring possible connections between tribology and mechanisms of synovial joint lubrication and degeneration (e.g., osteoarthritis) have been conducted by the author and his graduate and undergraduate students in the Department of Mechanical Engineering at Virginia Polytechnic Institute and State University. The basic approach used involved in vitro tribological experiments using bovine articular cartilage, with an emphasis on the effects of fluid composition and biochemistry on cartilage wear and damage. This research is an outgrowth of earlier work carried out during a sabbatical study in the Laboratory for the Study of Skeletal Disorders, The Children's Hospital Medical Center, Harvard Medical School in Boston. In that study, bovine cartilage test specimens were loaded against a polished steel plate and subjected to reciprocating sliding for several hours in the presence of a fluid (e.g., bovine synovial fluid or a buffered saline reference fluid containing biochemical constituents kindly provided by Dr. David Swann). Cartilage wear was determined by sampling the test fluid and determining the concentration of 4-hydroxyproline—a constituent of collagen. The results of that earlier study have been reported and summarized elsewhere [37-40]. Figure 4.4 shows the average hydroxyproline contents of wear debris obtained from these in vitro experiments. These numbers are related to the cartilage wear which occurred. However, since the total quantities of collected fluids varied somewhat, the values shown in the bar graph should not be taken as exact or precise measures of fluid effects on cartilage wear.

The main conclusions of that study were as follows:

1. Normal bovine synovial fluid is very effective in reducing cartilage wear under these in vitro conditions as compared to the buffered saline reference fluid.

2. There is no significant difference in wear between the saline reference and distilled water.

3. The addition of hyaluronic acid to the reference fluid significantly reduces wear, but its effect depends on the source.

4. Under these tests conditions, Swann's LGP-I (lubricating glycoprotein-I), known to be extremely effective in reducing friction in cartilage-on-glass tests, does not reduce cartilage wear.

Cartilage Wear
FIGURE 4.4 Relative cartilage wear based on hydroxyproline content of debris (in vitro tests with cartilage on stainless steel).

Synovial Fluid Constituents

Swann's LGP Glycoprotein

Reduce Cartilage Friction

*

-►

Protein Complex Hyaluronic Acid

Reduce Cartilage Wear

FIGURE 4.5 Friction and wear are different phenomena.

FIGURE 4.5 Friction and wear are different phenomena.

5. However, a protein complex isolated by Swann is extremely effective in reducing wear—producing results similar to those obtained with synovial fluid. The detailed structure of this constituent is complex and has not yet been fully determined.

6. Last, the lack of an added fluid in these experiments leads to extremely high wear and damage of the articular cartilage.

In discussing the possible significance of these findings from a tribological point of view, it may be helpful first of all to emphasize once again that friction and wear are different phenomena. Furthermore, as suggested by Fig. 4.5, certain constituents of synovial fluid (e.g., Swann's lubricating glycoprotein) may act to reduce friction in synovial joints while other constituents (e.g., Swann's protein complex or hyaluronic acid) may act to reduce cartilage wear. Therefore, it is necessary to distinguish between biochemical anti-friction and anti-wear compounds present in synovial fluid.

In more recent years, this study has been greatly enhanced by the participation of interested faculty and students from the Virginia-Maryland College of Veterinary Medicine and Department of Biochemistry and Animal Science at Virginia Tech. One major hypothesis tested is a continuation of previous work showing that the detailed biochemistry of the fluid-cartilage system has a pronounced and possibly controlling influence on cartilage wear. A consequence of the above hypothesis is that a lack or deficiency of certain biochemical constituents in the synovial joint may be one factor contributing to the initiation and progression of cartilage damage, wear, and possibly osteoarthritis. A related but somewhat different hypothesis concerns synovial fluid constituents which may act to increase the wear and further damage of articular cartilage under tribological contact.

To carry out continued research on biotribology, a new device for studies of cartilage deformation, wear, damage, and friction under conditions of tribological contact was designed by Burkhardt [33] and later modified, constructed, and instrumented. A simplified sketch is shown in Fig. 4.6. The key features of this test device are shown in Table 4.2. The apparatus is designed to accommodate cartilage-on-cartilage specimens. Motion of the lower specimen is controlled by a computer-driven X-Y table, allowing simple oscillating motion or complex motion patterns. An octagonal strain ring with two full semiconductor bridges is used to measure the normal load as well as the tangential load (friction). An LVDT, not shown in the figure, is used to measure cartilage deformation and linear wear during a test. However, hydroxyproline analysis of the wear debris and washings is used for the actual determination of total cartilage wear on a mass basis.

In one study by Schroeder [41], two types of experiments were carried out, i.e., cartilage-on-stainless steel and cartilage-on-cartilage, at applied loads up to 70 N—yielding an average pressure of 2.2 MPa in the contact area. Reciprocating motion (40 cps) was used. The fluids tested included: (1) a buffered saline solution, (2) saline plus hyaluronic acid, and (3) bovine synovial fluid. In cartilage-on-stainless steel tests, scanning electron microscopy, and histological staining showed distinct effects of the lubricants on surface and subsurface damage. Tests with the buffered saline fluid resulted in the most damage, with large wear tracks visible on the surface of the cartilage plug, as well as subsurface voids and cracks. When hyaluronic acid, a constituent of the natural synovial joint lubricant, was added to the saline reference fluid, less severe damage was observed. Little or no cartilage damage was evident in tests in which the natural synovial joint fluid was used as the lubricant.

Load

Cartilage Wear

FIGURE 4.6 Device for in vitro cartilage-on-cartilage wear studies.

TABLE 4.2 Key Features of Test Device Designed for Cartilage Wear Studies [33]

Contact system

Cartilage-on-cartilage

Contact geometry

Flat-on-flat, convex-on-flat, irregular-on-irregular

Cartilage type

Articular, any source (e.g., bovine)

Specimen size

Upper specimen, 4 to 6 mm diameter, lower specimen, ca. 15 to 25 mm diameter

Applied load

50-660 N

Average pressure

0.44-4.4 MPa

Type of motion

Linear, oscillating; circular, constant velocity; more complex patterns

Sliding velocity

0 to 20 mm/s

Fluid temperature

Ambient (20°C) or controlled humidity

Environment

Ambient or controlled humidity

Measurements

Normal load, cartilage deformation, friction; cartilage wear and damage, biochemical analysis of cartilage specimens, synovial fluid, and wear debris; sub-surface changes

These results were confirmed in a later study by Owellen [42] in which hydroxyproline analysis was used to determine cartilage wear. It was found that increasing the applied load from 20 to 65 N increased cartilage wear by eightfold for the saline solution and approximately threefold for synovial fluid. Furthermore, the coefficient of friction increased from an initial low value of 0.01 to 0.02 to a much higher value, e.g., 0.20 to 0.30 and higher, during a normal test which lasted 3 hours; the greatest change occurred during the first 20 minutes. Another interesting result was that a thin film of transferred or altered material was observed on the stainless steel disks—being most pronounced with the buffered saline lubricant and not observed with synovial fluid. Examination of the film with Fourier transfer infrared microspectrometry shows distinctive bio-organic spectra which differ from that of the original bovine cartilage. We believe this to be an important finding since it suggests a possible biotribochemical effect [43].

FIGURE 4.7 Cartilage damage produced by sliding contact.

In another phase of this research, the emphasis is on the cartilage-on-cartilage system and the influence of potentially beneficial as well as harmful constituents of synovial fluid on wear and damage. In cartilage-on-cartilage tests, the most severe wear and damage occurred during tests with buffered saline as the lubricant. The damage was less severe than in the stainless steel tests, but some visible wear tracks were detectable with scanning electron microscopy. Histological sectioning and staining of both the upper and lower cartilage samples show evidence of elongated lacunae and coalesced voids that could lead to wear by delamination. An example is shown in Fig. 4.7 (original magnification of 500x on 35-mm slide). The proteoglycan content of the subsurface cartilage under the region of contact was also reduced. When synovial fluid was used as the lubricant, no visible wear or damage was detected [44]. These results demonstrate that even in in vitro tests with bovine articular cartilage, the nature of the fluid environment can have a dramatic affect on the severity of wear and subsurface damage.

In a more recent study carried out by Berrien in the biotribology program at Virginia Tech, a different approach was taken to examine the role of joint lubrication in joint disease, particularly osteoarthritis. A degradative biological enzyme, collagenase-3, suspected of playing a role in a cartilage degeneration was used to create a physiologically adverse biochemical fluid environment. Tribological tests were performed with the same device and procedures described previously. The stainless steel disk was replaced with a 1-in. diameter plug of bovine cartilage to create a cartilage sliding on cartilage configuration more closely related to the in vivo condition. Normal load was increased to 78.6 N and synovial fluid and buffered saline were used as lubricants. Prior to testing, cartilage plugs were exposed to a fluid medium containing three concentrations of collagenase-3 for 24 h. The major discovery of this work was that exposure to the collagenase-3 enzyme had a substantial adverse effect on cartilage wear in vitro, increasing average wear values by three and one-half times those of the unexposed cases. Figure 4.8 shows an example of the effect of enzyme treatment when bovine synovial fluid was used as the lubricant. Scanning electron microscopy showed disruption of the superficial layer and collagen matrix with exposure to collagenase-3, where unexposed cartilage showed none. Histological sections showed a substantial loss of the superficial layer of cartilage and a distinct and abnormal loss of proteoglycans in the middle layer of collagenase-treated cartilage. Unexposed cartilage showed only minor disruption of the superficial layer [45].

This study indicates that some of the biochemical constituents that gain access to the joint space, during normal and pathological functions, can have a significant adverse effect on the wear and damage o> E

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